Tools and methods for expression of membrane proteins

ABSTRACT

The disclosure relates cells or cellular systems that express both a membrane protein and a binding domain directed to the membrane protein. Also, methods are provided that use such cells or cellular systems to produce higher amounts of the membrane proteins. Further, the cells or cellular systems can be used as tools for the structural and functional characterization of membrane proteins, as well as for screening and drug discovery efforts targeting membrane proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/373,329, filed Jul. 18, 2014, which will issue as U.S. Pat. No.9,890,217 on Feb. 13, 2018, which is a national phase entry under 35U.S.C. § 371 of International Patent Application PCT/EP2013/051041,filed Jan. 21, 2013, designating the United States of America andpublished in English as International Patent Publication WO2013/107905A1 on Jul. 25, 2013, which claims the benefit under Article 8 of thePatent Cooperation Treaty to European Application Serial No. 12151814.6,filed Jan. 19, 2012, and under 35 U.S.C. § 119(e) to U.S. ProvisionalApplication Ser. No. 61/588,523, filed Jan. 19, 2012, the disclosure ofeach of which is hereby incorporated herein in its entirety by thisreference.

STATEMENT ACCORDING TO 37 C.F.R. § 1.821(c) or (e)—SEQUENCE LISTINGSUBMITTED AS ASCII TEXT FILE

Pursuant to 37 C.F.R. § 1.821(c) or (e), a file containing an ASCII textversion of the Sequence Listing has been submitted concomitant with thisapplication, the contents of which are hereby incorporated by reference.

TECHNICAL FIELD

The disclosure relates to the field of protein expression technologies.More specifically, cells or cellular systems are provided that expressboth a membrane protein and a binding domain directed to the membraneprotein. Also, methods are provided that use such cells or cellularsystems to produce higher amounts of the membrane proteins. Further, thecells or cellular systems can be used as tools for the structural andfunctional characterization of membrane proteins, as well as forscreening and drug discovery efforts targeting membrane proteins.

BACKGROUND

There has been increasing interest and progress in the field of membraneprotein research over the last years. The high interest in membraneproteins is due to the fact that they play an important role in severalbiological processes such as ion transport, recognition of molecules,signal transduction, etc. G-protein coupled receptors (GPCRs) constitutethe largest family of transmembrane proteins and play an important partin signal transduction by converting extracellular stimuli includinglight, smells, neurotransmitters and hormones, into intracellularsignals. Currently, more than 30% of marketed medicines act on GPCRs,which are considered as attractive targets for new medicines.Structure-based drug discovery and high throughput screening (HTS) fornovel compounds active on a receptor of interest have now become anintegrated technology in pharmaceutical laboratories.

Although membrane proteins represent 20% to 30% of all genes inprokaryotes as well as in eukaryotes, only little is known aboutstructure and function relationship of membrane proteins. This can belargely attributed to the low natural expression of membrane proteins,to their hydrophobic character, which complicates overexpression offunctional membrane proteins, as well as to difficulties during theirpurification and crystallization. By way of example, only for sevenGPCRs high-resolution structures have been characterized: rhodopsin, theβ1 and β2 adrenergic receptors, the adenosine 2A receptor, and morerecently the CXCR4 receptor, the dopamine D3 receptor and the histamineH1 receptor. Whereas rhodopsin was purified and subsequentlycrystallized from unmodified protein isolated from native tissue (a loneexception to the rule of low expression levels), these other GPCRsrequired expression in recombinant systems, stabilization of an inactivestate by an inverse agonist/antagonist and biochemical modifications tostabilize the receptor protein (e.g., Rasmussen et al., 2007, Nature450:383; Rosenbaum et al., 2007, Science, 318:1266; Warne et al., 2008,Nature 454:486; Wu et al., 2010, Science 330:1066; Chien et al., 2010,Science 330:091; Shimamura et al., 2011, Nature 475: 65. Up till now,most unravelled structures have the third cytoplasmic loop replaced forthe very stable bacteriophage T4 lysozyme. This fusion protein mighthowever not represent the true natural conformation of the GPCR.Therefore, the structures need to be analyzed with great care whenperforming ligand screening and drug design. Besides that, evidence fromfunctional and biophysical studies shows that GPCRs can exist inmultiple functionally distinct conformational states (Kobilka and Deupi2007, Trends Pharmacol Sciences 28:397). While this structuralplasticity and dynamic behavior is essential for normal function, itcontributes to their biochemical instability and difficulty in obtaininghigh-resolution crystal structures. Only recently it became possible toobtain structures of an active state of a GPCR, making use ofstabilizing Nanobodies (Rasmussen et al., 2011, Nature 469: 175).

Most membrane proteins express at low levels in non-engineeredeukaryotic cells. Eukaryotic membrane proteins have been successfullyoverexpressed in bacteria, yeast, mammalian cell lines and insect cells(reviewed in Freigassner et al., 2009, Microb Cell Fact. 8:69). However,expression levels are still rather low and for the majority of thesereceptors a 5- to 10-fold increase in expression level would lead tosufficient material for subsequent experiments, especially proteinpurification and characterization, structural and pharmacologicalstudies. For example, expression of eukaryotic membrane proteins inprokaryotic systems mostly leads to poor expression levels. Besides, inmany cases the protein ends up in denatured form in inclusion bodies andthe necessity of complicated refolding processes has hampered thesuccess. Expression in yeast is a valuable alternative for theexpression of eukaryotic membrane proteins. Yeast cells are easy tohandle, and can grow in fermentors to very high cell densities.Different techniques to increase the expression levels of the membraneprotein have been used in yeast such as lowering the inductiontemperature, adding antagonist, DMSO or histidine to the inductionmedium (André et al., 2006, Protein Sci. 15:1115). Other approaches havebeen taken to enhance membrane protein surface expression inheterologous cells, including addition/deletion of receptor sequences,co-expression with interacting proteins, and treatment withpharmacological chaperones (reviewed in Dunham and Hall, 2009, TrendsBiotechnol. 27:541). It remains a challenge, however, to significantlyimprove total yield, conformational stability and/or functionality ofwild-type surface expressed membrane protein.

Thus, it would be advantageous to have alternative expression systemsthat permit higher heterologous expression of native membrane proteinsin a particular conformation. This would greatly facilitate the wholetrajectory of drug discovery efforts on membrane proteins as therapeutictargets.

SUMMARY OF THE INVENTION

The disclosure provides tools and methods for heterologous expression ofmembrane proteins which are of particular advantage. To illustrate this,without being limitative, the human CXCR4 (hCXCR4) GPCR was co-expressedwith Nanobodies (Nbs) directed against hCXCR4 in the yeast strain P.pastoris. It was surprisingly found that hCXCR4-Nb co-expression resultsin an increase in expression of the membrane protein, as compared tohCXCR4 expression alone. In addition, because of the interaction betweenthe GPCR and the Nb, the membrane protein can be directly purified fromthe co-expressing host cell using affinity-based purification methods.Furthermore, when a conformation-selective Nb is used, it is possible topurify the fraction of the membrane proteins that particularly residesin the conformation as stabilized by the Nb. Thus, using the tools andmethods of the disclosure, the homogeneity of a membrane protein sampleis increased, which is particularly useful, e.g., for subsequentcrystallization, immunization or compound screening. The skilled artisanwill understand that the above findings will generally be applicable forthe co-expression of other type of membrane proteins and binding domainsdirected against these membrane proteins.

Accordingly, a first aspect of the disclosure relates to a host cellcomprising a first exogenous nucleic acid sequence encoding a membraneprotein and a second exogenous nucleic acid encoding a binding domaindirected against the membrane protein, each under the control of apromoter. In a preferred embodiment, the promoter is an induciblepromoter. In another preferred embodiment, the membrane protein andbinding domain are co-expressed. In still another embodiment, themembrane protein and/or the binding domain are operably linked to asubcellular targeting sequence, such as an ER or Golgi localizationsignal or secretion signal.

According to particular embodiments, the membrane protein in any of theabove-described host cells is a membrane receptor protein, such as aGPCR, or a membrane transport protein, such as an ion transporter.

A further embodiment of the disclosure relates to any of theabove-described host cells wherein the binding domain specifically bindsto an extracellular conformational epitope of the membrane protein.

Preferably, the binding domain in any of the above-described host cellsis an immunoglobulin single variable domain comprising an amino acidsequence comprising 4 framework regions and 3complementarity-determining regions, or any suitable fragment thereof.More preferably, the immunoglobulin single variable domain is a VHH.

In a particularly preferred embodiment of any of the above-describedhost cells, the immunoglobulin single variable domain stabilizes themembrane protein in a functional conformational state, such as an activeor an inactive state.

The host cell, according to the disclosure, may be a eukaryotic hostcell, such as a yeast cell, a mammalian cell, an insect cell. Inparticular, the yeast may be a Pichia strain, such as a Pichia pastoris,or a Komagataella strain, such as Komagataella pastoris, or a Hansenulastrain, such as Hansenula polymorpha, or a Yarrowia strain, such asYarrowia lipolytica, or a Saccharomyces strain, such as Saccharomycescerevisiae, and wherein the filamentous fungi is an Aspergillus strain,such as Aspergillus niger or Aspergillus nidulans, or a Penicilliumstrain, such as Penicillium citrinum or Penicillium chrysogenum, or aHypocrea strain, such as Hypocrea jecorina.

In addition, the host cell may be a glycoengineered host cell.

In another aspect, the disclosure also envisages cell cultures of any ofthe above-described host cells or membrane preparations derived of thehost cells or cell cultures. Expression vectors comprising the firstand/or the second exogenous nucleic acid sequence as comprised in thehost cells according to the disclosure are also encompassed.

According to a further aspect, the disclosure relates to a method ofenhancing the production of a membrane protein in a host cell comprisingthe steps of:

-   -   a. Providing a host cell as described above,    -   b. Culturing the host cell under conditions suitable for        co-expressing the membrane protein and the binding domain        directed against the membrane protein.

Also envisages is a method of enhancing the production of a membraneprotein in a functional conformation in a host cell, the methodcomprising the steps of:

-   -   a. Providing a host cell as described above,    -   b. Culturing the host cell under conditions suitable for        co-expressing the membrane protein and the binding domain        directed against the membrane protein, in the presence of a        conformation-selective ligand, such as an agonist, an        antagonist, an inverse agonist.

The method may further comprise the step of isolating the membraneprotein, the binding domain or the membrane protein in complex with thebinding domain.

Finally, the disclosure also relates to the use of the host cells, orthe cell cultures, or the membrane preparations, or the proteinsisolated therefrom, all as described hereinbefore, for ligandcharacterization, drug screening, protein capturing and purification,immunization, biophysical studies, amongst others. Other applicationswill become clear from the description further herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Protein alignment of the four anti-hCXCR4 Nanobody sequences.The level of conservation between the four different anti-hCXCR4Nanobodies is graphically depicted as a line plot and a sequence logo(alignment was done using the CLC DNA Workbench software).

FIG. 2: Schematic overview of the pKai61-anti-hCXCR4Nb vector containingthe anti-hCXCR4Nb gene under control of the AOX1 promoter. This vectorwas linearized with restriction enzyme PmeI to facilitate targetedintegration in the AOX1 locus of the Pichia genome.

FIGS. 3A and 3B: FIG. 3A: SDS-PAGE Coomassie Brilliant Blue detection ofthe four anti-hCXCR4 Nanobodies. M=All blue precision plus proteinstandards. FIG. 3B: immunoblot detection of the four anti-hCXCR4Nanobodies. Mouse anti-His antibody and secondary goat anti-mouse (800nm) DyLight antibody was used for the detection of the nanobodies on theOdyssey system.

FIGS. 4A and 4B: FIG. 4A: Protein sequence of hCXCR4Rho1D4 fused to thealpha-mating factor of S. cerevisiae. Schematic overview of thepPIC92hCXCR4Rho1D4 expression vector, containing the hCXCR4Rho1D4 geneunder control of the AOX1 promoter (SEQ ID NO:32). FIG. 4B: vectorlinearized with restriction enzyme StuI to facilitate targetedintegration in the HIS4 locus of the Pichia genome.

FIG. 5: Immunoblot analysis of hCXCR4Rho1D4 expressed in P. pastoris.M=All blue precision plus protein standards. Mouse anti-Rho1D4 antibodyand secondary goat anti-mouse (800 nm) DyLight antibody was used for thedetection of hCXCR4Rho1D4 on the Odyssey system.

FIG. 6: Immunoblot analysis of hCXCR4Rho1D4 expressed in P. pastoriscells, expressing only hCXCR4Rho1D4 or co-expressing hCXCR4Rho1D4 and ananti-hCXCR4 Nanobody. M=All blue precision plus protein standards. Mouseanti-Rho1D4 antibody and secondary goat anti-mouse (800 nm) DyLightantibody was used for the detection of hCXCR4Rho1D4 on the Odysseysystem.

FIGS. 7A and 7B: Immunoblot analysis showing intracellular retention ofthe anti-hCXCR4 Nanobodies. FIG. 7A: Nanobody CA4140 and CA4142 both inthe presence or absence of hCXCR4Rho1D4. FIG. 7B: Nanobody CA4143 andCA4500 both in the presence or absence of hCXCR4Rho1D4. M=All blueprecision plus protein standards. Mouse anti-His antibody and secondarygoat anti-mouse (800 nm) DyLight antibody was used for the detection ofthe nanobodies on the Odyssey system.

FIG. 8: Immunoblot analysis showing no increase in expression of thehCXCR4 receptor upon co-expression of a Nanobody that does not recognizethe receptor (NbCA4910).M=All blue precision plus protein standards.Mouse anti-Rho1D4 antibody and secondary goat anti-mouse (800 nm)DyLight antibody was used for the detection of hCXCR4Rho1D4 on theOdyssey system.

FIG. 9: Immunoblot analysis showing different purification steps of thehCXCR4 receptor using the Nanobody CA4142 as a purification handle.Respectively, the lanes contain cell extract, flowthrough, wash fraction1, 2 and 3, M, elution 1 (100 mM Imidazole), elution 2, 3 and 4 (using400 mM Imidazole). M=All blue precision plus protein standards. Mouseanti-Rho1D4 antibody and secondary goat anti-mouse (800 nm) DyLightantibody was used for the detection of the receptor on the Odysseysystem.

FIG. 10: Immunoblot analysis showing different purification steps of theb2AR using Nanobody CA2780 as a purification handle. Respectively, thelanes contain flowthrough, wash fraction 1 and 2, M, elution 1 (100 mMImidazole), elution 2 (200 mM Imidazole), 3 and 4 (using 400 mMImidazole). M=All blue precision plus protein standards. Mouseanti-Rho1D4 antibody and secondary goat anti-mouse (680 nm) DyLightantibody was used for the detection of the receptor, and 6×His TagAntibody Dylight 800 antibody was used for the detection of the Nanobodyon the Odyssey system.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure will be described with respect to particular embodimentsand with reference to certain drawings, but the disclosure is notlimited thereto but only by the claims. Any reference signs in theclaims shall not be construed as limiting the scope. The drawingsdescribed are only schematic and are non-limiting. In the drawings, thesize of some of the elements may be exaggerated and not drawn on scalefor illustrative purposes. Where the term “comprising” is used in thedescription and claims, it does not exclude other elements or steps.Where an indefinite or definite article is used when referring to asingular noun, e.g., “a” or “an,” “the,” this includes a plural of thatnoun unless something else is specifically stated. Furthermore, theterms first, second, third and the like in the description and in theclaims, are used for distinguishing between similar elements and notnecessarily for describing a sequential or chronological order. It is tobe understood that the terms so used are interchangeable underappropriate circumstances and that the embodiments of the disclosuredescribed herein are capable of operation in other sequences thandescribed or illustrated herein.

Unless otherwise defined herein, scientific and technical terms andphrases used in connection with the disclosure shall have the meaningsthat are commonly understood by those of ordinary skill in the art.Generally, nomenclatures used in connection with, and techniques ofmolecular and cellular biology, genetics and protein and nucleic acidchemistry and hybridization, described herein, are those well-known andcommonly used in the art. The methods and techniques of the disclosureare generally performed according to conventional methods well known inthe art and as described in various general and more specific referencesthat are cited and discussed throughout the specification unlessotherwise indicated. See, for example, Sambrook et al. MolecularCloning: A Laboratory Manual, 2d ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989); Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates (1992, andSupplements to 2002).

Definitions

The term “membrane protein,” as used herein, refers to a protein that isattached to or associated with a membrane of a cell or an organelle.Specific non-limiting examples are provided further in thespecification.

The term “protein binding domain” or simply “binding domain” refersgenerally to any non-naturally occurring molecule or part thereof thatis able to bind to a protein or peptide using specific intermolecularinteractions. A variety of molecules can function as protein bindingdomains, including, but not limited to, proteinaceous molecules(protein, peptide, protein-like or protein containing), nucleic acidmolecules (nucleic acid, nucleic acid-like, nucleic acid containing),and carbohydrate molecules (carbohydrate, carbohydrate-like,carbohydrate containing). A more detailed description can be foundfurther in the specification.

As used herein, the terms “polypeptide,” “protein,” “peptide” are usedinterchangeably herein, and refer to a polymeric form of amino acids ofany length, which can include coded and non-coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones.

As used herein, the terms “nucleic acid molecule,” “polynucleotide,”“polynucleic acid,” “nucleic acid” are used interchangeably and refer toa polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three-dimensional structure, and mayperform any function, known or unknown. Non-limiting examples ofpolynucleotides include a gene, a gene fragment, exons, introns,messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, control regions, isolated RNA ofany sequence, nucleic acid probes, and primers. The nucleic acidmolecule may be linear or circular.

The term “conformation” or “conformational state” of a protein refersgenerally to the range of structures that a protein may adopt at anyinstant in time. One of skill in the art will recognize thatdeterminants of conformation or conformational state include a protein'sprimary structure as reflected in a protein's amino acid sequence(including modified amino acids) and the environment surrounding theprotein. The conformation or conformational state of a protein alsorelates to structural features such as protein secondary structures(e.g., α-helix, β-sheet, among others), tertiary structure (e.g., thethree-dimensional folding of a polypeptide chain), and quaternarystructure (e.g., interactions of a polypeptide chain with other proteinsubunits). Post-translational and other modifications to a polypeptidechain such as ligand binding, phosphorylation, sulfation, glycosylation,or attachments of hydrophobic groups, among others, can influence theconformation of a protein. Furthermore, environmental factors, such aspH, salt concentration, ionic strength, and osmolality of thesurrounding solution, and interaction with other proteins andco-factors, among others, can affect protein conformation. Theconformational state of a protein may be determined by either functionalassay for activity or binding to another molecule or by means ofphysical methods such as X-ray crystallography, NMR, or spin labeling,among other methods. For a general discussion of protein conformationand conformational states, one is referred to Cantor and Schimmel,Biophysical Chemistry, Part I: The Conformation of Biological.Macromolecules, W.H. Freeman and Company, 1980, and Creighton, Proteins:Structures and Molecular Properties, W.H. Freeman and Company, 1993. A“specific conformational state” is any subset of the range ofconformations or conformational states that a protein may adopt.

A “functional conformation” or a “functional conformational state,” asused herein, refers to the fact that proteins possess differentconformational states having a dynamic range of activity, in particularranging from no activity to maximal activity. It should be clear that “afunctional conformational state” is meant to cover any conformationalstate of a protein, in particular a membrane protein, having anyactivity, including no activity; and is not meant to cover the denaturedstates of proteins. A particular class of functional conformations isdefined as “drugable conformation” and generally refers to a uniquetherapeutically relevant conformational state of a target protein. As anillustration, the active conformation of the β2 adrenergic receptorcorresponds to the drugable conformation of this receptor for thetreatment of asthma. It will thus be understood that drugability isconfined to particular conformations depending on the therapeuticindication.

As used herein, the terms “complementarity determining region” or “CDR”within the context of antibodies refer to variable regions of either H(heavy) or L (light) chains (also abbreviated as VH and VL,respectively) and contains the amino acid sequences capable ofspecifically binding to antigenic targets. These CDR regions account forthe basic specificity of the antibody for a particular antigenicdeterminant structure. Such regions are also referred to as“hypervariable regions.” The CDRs represent non-contiguous stretches ofamino acids within the variable regions but, regardless of species, thepositional locations of these critical amino acid sequences within thevariable heavy and light chain regions have been found to have similarlocations within the amino acid sequences of the variable chains. Thevariable heavy and light chains of all canonical antibodies each have 3CDR regions, each non-contiguous with the others (termed L1, L2, L3, H1,H2, H3) for the respective light (L) and heavy (H) chains. Nanobodies,in particular, generally comprise a single amino acid chain that can beconsidered to comprise 4 “framework sequences or regions” or FRs and 3“complementarity-determining regions” or CDRs. The nanobodies have 3 CDRregions, each non-contiguous with the others (termed CDR1, CDR2, CDR3).The delineation of the FR and CDR sequences is based on the IMGT uniquenumbering system for V-domains and V-like domains (Lefranc et al., 2003,Developmental and Comparative Immunology 27:55).

An “epitope,” as used herein, refers to an antigenic determinant of apolypeptide. An epitope could comprise 3 amino acids in a spatialconformation, which is unique to the epitope. Generally, an epitopeconsists of at least 4, 5, 6, 7 such amino acids, and more usually,consists of at least 8, 9, 10 such amino acids. Methods of determiningthe spatial conformation of amino acids are known in the art, andinclude, for example, x-ray crystallography and two-dimensional nuclearmagnetic resonance.

A “conformational epitope,” as used herein, refers to an epitopecomprising amino acids in a spatial conformation that is unique to afolded three-dimensional conformation of a polypeptide. Generally, aconformational epitope consists of amino acids that are discontinuous inthe linear sequence that come together in the folded structure of theprotein. However, a conformational epitope may also consist of a linearsequence of amino acids that adopts a conformation that is unique to afolded three-dimensional conformation of the polypeptide (and notpresent in a denatured state). In multiprotein complexes, conformationalepitopes consist of amino acids that are discontinuous in the linearsequences of one or more polypeptides that come together upon folding ofthe different folded polypeptides and their association in a uniquequaternary structure.

The term “specificity,” as used herein, refers to the ability of abinding domain, in particular an immunoglobulin or an immunoglobulinfragment, such as a VHH or nanobody, to bind preferentially to oneantigen, versus a different antigen, and does not necessarily imply highaffinity.

The term “affinity,” as used herein, refers to the degree to which abinding domain, in particular an immunoglobulin, such as an antibody, oran immunoglobulin fragment, such as a VHH or nanobody, binds to anantigen so as to shift the equilibrium of antigen and protein bindingdomain toward the presence of a complex formed by their binding. Thus,for example, where an antigen and antibody (fragment) are combined inrelatively equal concentration, an antibody (fragment) of high affinitywill bind to the available antigen so as to shift the equilibrium towardhigh concentration of the resulting complex. The dissociation constantis commonly used to describe the affinity between the protein bindingdomain and the antigenic target. Typically, the dissociation constant islower than 10⁻⁵ M. Preferably, the dissociation constant is lower than10⁻⁶ M, more preferably, lower than 10⁻⁷ M. Most preferably, thedissociation constant is lower than 10⁻⁸ M.

The terms “specifically bind” and “specific binding,” as used herein,generally refers to the ability of a binding domain, in particular animmunoglobulin, such as an antibody, or an immunoglobulin fragment, suchas a VHH or nanobody, to preferentially bind to a particular antigenthat is present in a homogeneous mixture of different antigens. Incertain embodiments, a specific binding interaction will discriminatebetween desirable and undesirable antigens in a sample, in someembodiments more than about 10- to 100-fold or more (e.g., more thanabout 1000- or 10,000-fold). Within the context of the spectrum ofconformational states of GPCRs, the terms particularly refer to theability of a binding domain (as defined herein) to preferentiallyrecognize and/or bind to a particular conformational state of a GPCR ascompared to another conformational state. For example, an activestate-selective protein binding domain will preferentially bind to aGPCR in an active conformational state and will not or to a lesserdegree bind to a GPCR in an inactive conformational state, and will thushave a higher affinity for the active conformational state. The terms“specifically bind,” “selectively bind,” “preferentially bind,” andgrammatical equivalents thereof, are used interchangeably herein. Theterms “conformational specific” or “conformational selective” are alsoused interchangeably herein.

A “deletion” is defined here as a change in either amino acid ornucleotide sequence in which one or more amino acid or nucleotideresidues, respectively, are absent as compared to an amino acid sequenceor nucleotide sequence of a parental polypeptide or nucleic acid. Withinthe context of a protein, a deletion can involve deletion of about 2,about 5, about 10, up to about 20, up to about 30 or up to about 50 ormore amino acids. A protein or a fragment thereof may contain more thanone deletion. Within the context of a GPCR, a deletion may also be aloop deletion, or an N- and/or C-terminal deletion.

An “insertion” or “addition” is that change in an amino acid ornucleotide sequences, which has resulted in the addition of one or moreamino acid or nucleotide residues, respectively, as compared to an aminoacid sequence or nucleotide sequence of a parental protein. “Insertion”generally refers to addition to one or more amino acid residues withinan amino acid sequence of a polypeptide, while “addition” can be aninsertion or refer to amino acid residues added at an N- or C-terminus,or both termini. Within the context of a protein or a fragment thereof,an insertion or addition is usually of about 1, about 3, about 5, about10, up to about 20, up to about 30 or up to about 50 or more aminoacids. A protein or fragment thereof may contain more than oneinsertion.

A “substitution,” as used herein, results from the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively, as compared to an amino acid sequence or nucleotidesequence of a parental protein or a fragment thereof. It is understoodthat a protein or a fragment thereof may have conservative amino acidsubstitutions which have substantially no effect on the protein'sactivity. By conservative substitutions is intended combinations such asgly, ala; val, ile, leu, met; asp, glu; asn, gln; ser, thr; lys, arg;cys, met; and phe, tyr, trp.

The term “compound” or “test compound” or “candidate compound” or “drugcandidate compound,” as used herein, describes any molecule, eithernaturally occurring or synthetic that is tested in an assay, such as ascreening assay or drug discovery assay. As such, these compoundscomprise organic or inorganic compounds. The compounds includepolynucleotides, lipids or hormone analogs that are characterized by lowmolecular weights. Other biopolymeric organic test compounds includesmall peptides or peptide-like molecules (peptidomimetics) comprisingfrom about 2 to about 40 amino acids and larger polypeptides comprisingfrom about 40 to about 500 amino acids, such as antibodies, antibodyfragments or antibody conjugates. Test compounds can also be proteinscaffolds. For high-throughput purposes, test compound libraries may beused, such as combinatorial or randomized libraries that provide asufficient range of diversity. Examples include, but are not limited to,natural compound libraries, allosteric compound libraries, peptidelibraries, antibody fragment libraries, synthetic compound libraries,fragment-based libraries, phage-display libraries, and the like. A moredetailed description can be found further in the specification.

As used herein, the term “ligand” means a molecule that specificallybinds to a membrane protein, either intracellularly or extracellularly.A ligand may be, without the purpose of being limitative, a protein, a(poly)peptide, a lipid, a small molecule, a protein scaffold, a nucleicacid, an ion, a carbohydrate, an antibody or an antibody fragment, suchas a nanobody (all as defined herein). A ligand may be synthetic ornaturally occurring. A ligand also includes a “native ligand,” which isa ligand that is an endogenous, natural ligand for a native protein.Usually, a membrane protein will adopt a particular conformation uponbinding of a ligand. Thus, a ligand is also referred to herein as a“conformation-selective ligand” or “conformation-specific ligand.” Theterm includes agonists, full agonists, partial agonists, inverseagonists, and antagonists, binding at either the orthosteric site or atan allosteric site.

An “orthosteric ligand,” as used herein, refers to a ligand (bothnatural and synthetic), that they binds to the active site of a membraneprotein, in particular a receptor protein, such as a GPCR, and arefurther classified according to their efficacy or in other words to theeffect they have on signaling through a specific pathway. As usedherein, an “agonist” refers to a ligand that, by binding a membraneprotein, increases the membrane protein's signaling activity. Fullagonists are capable of maximal protein stimulation; partial agonistsare unable to elicit full activity even at saturating concentrations.Partial agonists can also function as “blockers” by preventing thebinding of more robust agonists. An “antagonist” refers to a ligand thatbinds a membrane protein without stimulating any activity. An“antagonist” is also known as a “blocker” because of its ability toprevent binding of other ligands and, therefore, block agonist-inducedactivity. Further, an “inverse agonist” refers to an antagonist that, inaddition to blocking agonist effects, reduces a membrane proteins' basalor constitutive activity below that of the unliganded protein.

Ligands, as used herein, may also be “biased ligands” with the abilityto selectively stimulate a subset of a membrane protein's signalingactivities, for example, in the case of GPCRs the selective activationof G-protein or β-arrestin function. Such ligands are known as “biasedligands,” “biased agonists” or “functionally selective agonists.” Moreparticularly, ligand bias can be an imperfect bias characterized by aligand stimulation of multiple membrane protein activities withdifferent relative efficacies for different signals (non-absoluteselectivity) or can be a perfect bias characterized by a ligandstimulation of one membrane protein activity without any stimulation ofanother known membrane protein activity.

Another kind of ligands is known as allosteric regulators. “Allostericregulators” or otherwise “allosteric modulators,” “allosteric ligands”or “effector molecules,” as used herein, refer to ligands that bind atan allosteric site (that is, a regulatory site physically distinct fromthe protein's active site) of a membrane protein, in particular areceptor protein such as a GPCR. In contrast to orthosteric ligands,allosteric modulators are non-competitive because they bind membraneproteins at a different site and modify their function even if theendogenous ligand also is binding. Allosteric regulators that enhancethe protein's activity are referred to herein as “allosteric activators”or “positive allosteric modulators,” whereas those that decrease theprotein's activity are referred to herein as “allosteric inhibitors” orotherwise “negative allosteric modulators.”

As used herein, the terms “determining,” “measuring,” “assessing,”“monitoring” and “assaying” are used interchangeably and include bothquantitative and qualitative determinations.

The term endogenous,” as used herein, refers to substances (e.g., genes)originating from within an organism, tissue, or cell. Analogously,“exogenous,” as used herein, is any material that comes from outside anorganism, tissue, or cell, but that is present (and typically can becomeactive) in that organism, tissue, or cell.

The term “inducible promoter,” as used herein, refers to a promoter thatcan be switched “on” or “off” (thereby regulating gene transcription) inresponse to external stimuli such as, but not limited to, temperature,pH, certain nutrients, specific cellular signals, etcetera. It is usedto distinguish between a “constitutive promoter,” by which a promoter ismeant that is continuously switched “on,” i.e., from which genetranscription is constitutively active.

The term “subcellular targeting sequence,” as used herein, generallyrefers to a molecule that directs localization of proteins to differentcell compartments. Examples include an “ER localization signal” or a“Golgi localization signal,” which is a molecule, typically a peptidethat directs localization of the polypeptide or protein to which it isconjugated to the ER or Golgi apparatus, respectively. Localization,thus, also implies retention in the ER or Golgi apparatus, respectively.Typically, these localization (or retention) sequences are peptidesequences derived from (pre)proteins that are situated in the ER orGolgi when functionally active as a mature protein and examples areprovided further herein. In particular, the C-terminal addition of thetetrapeptide H/KDEL to a soluble protein that is translocated in the ERcan be used to retain this protein in the ER. Subcellular targetingsequences also include secretion signals of which examples are alsoprovided further herein.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid molecule towhich it has been linked. One type of vector is a “plasmid vector,”which refers to a circular double stranded DNA loop into whichadditional DNA segments may be ligated. Other vectors include, withoutthe purpose of being limitative, cosmids and yeast artificialchromosomes (YAC). Certain vectors are capable of autonomous replicationin a host cell into which they are introduced (e.g., vectors having anorigin of replication which functions in the host cell). Other vectorscan be integrated into the genome of a host cell upon introduction intothe host cell, and are thereby replicated along with the host genome.Moreover, certain preferred vectors are capable of directing theexpression of certain genes of interest. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “expressionvectors”). Suitable vectors have regulatory sequences, such aspromoters, enhancers, terminator sequences, and the like as desired.

The term “regulatory sequence,” as used herein, refers to polynucleotidesequences, which are necessary to affect the expression of codingsequences to which they are operably linked. Expression controlsequences are sequences which control the transcription,post-transcriptional events and translation of nucleic acid sequences.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRMA; sequences that enhancetranslation efficiency (e.g., ribosome binding sites); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism. The term “control sequences” isintended to include, at a minimum, all components whose presence isessential for expression, and can also include additional componentswhose presence is advantageous, for example, leader sequences and fusionpartner sequences.

The term “operably linked,” as used herein, refers to a linkage in whichthe regulatory sequence is contiguous with the gene of interest tocontrol the gene of interest, as well as regulatory sequences that actin trans or at a distance to control the gene of interest.

The term “host cell” and equivalent terms like “recombinant host cell,”“expression host cell,” “expression host system,” “expression system,”as used herein, is intended to refer to a cell into which a recombinantvector has been introduced. It should be understood that such terms areintended to refer not only to the particular subject cell but to theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell,”as used herein. A recombinant host cell may be an isolated cell or cellline grown in culture or may be a cell which resides in a living tissueor organism. Particular examples are provided further herein.

Detailed Description

The disclosure provides engineered cells or cellular systems (cellcultures, organisms) that can express proteins, particularly membraneproteins, at higher levels either at the cellular surface or in othercell compartments. In particular, the proteins that are expressed bythese cells or cellular systems are maintained or stabilized in aparticular functional conformation. Further, methods are provided thatuse such cells or cellular systems to produce these proteins. It will beappreciated that while the disclosure has been exemplified with GPCRs,it is equally applicable to any membrane protein, especially a membraneprotein that is poorly expressed and has a low stability in arecombinant host cell.

Accordingly, a first aspect of the disclosure relates to a host cellcomprising a first exogenous nucleic acid sequence encoding a membraneprotein and a second exogenous nucleic acid encoding a binding domaindirected against the membrane protein, each under the control of apromoter.

The “host cell,” according to the disclosure, can be of any prokaryoticor eukaryotic organism. According to a preferred embodiment, the hostcell is a eukaryotic cell and can be of any eukaryotic organism, but inparticular embodiments yeast, plant, mammalian and insect cells areenvisaged. The nature of the cells used will typically depend on theease and cost of producing the native protein(s), the desiredglycosylation properties, the origin of the target protein, the intendedapplication, or any combination thereof. Mammalian cells may, forinstance, be used for achieving complex glycosylation, but it may not becost-effective to produce proteins in mammalian cell systems. Plant andinsect cells, as well as yeast typically achieve high production levelsand are more cost-effective, but additional modifications may be neededto mimic the complex glycosylation patterns of mammalian proteins or toavoid excessive glycosylation heterogeneity which might hamper crystalformation or structural determination. Yeast cells are currentlypreferred for expression of proteins because they can be economicallycultured, give high yields of protein, and when appropriately modifiedare capable of producing proteins having suitable glycosylationpatterns. Further, yeast offers established genetics allowing for rapidtransformations, tested protein localization strategies, and facile geneknock-out techniques. Eukaryotic cell or cell lines for proteinproduction are well known in the art, including cell lines with modifiedglycosylation pathways, and non-limiting examples will be providedhereafter.

Animal or mammalian host cells suitable for harboring, expressing, andproducing proteins for subsequent isolation and/or purification may befrom human or non-human origin, and include Chinese hamster ovary cells(CHO), such as CHO-K1 (ATCC CCL-61), DG44 (Chasin et al., 1986, Som.Cell Molec. Genet., 12:555-556; and Kolkekar et al., 1997, Biochemistry,36:10901-10909), CHO-K1 Tet-On cell line (Clontech), CHO designatedECACC 85050302 (CAMR, Salisbury, Wiltshire, UK), CHO clone 13 (GEIMG,Genova, IT), CHO clone B (GEIMG, Genova, IT), CHO-K1/SF designated ECACC93061607 (CAMR, Salisbury, Wiltshire, UK), RR-CHOK1 designated ECACC92052129 (CAMR, Salisbury, Wiltshire, UK), dihydrofolate reductasenegative CHO cells (CHO/−DHFR, Urlaub and Chasin, 1980, Proc. Natl.Acad. Sci. USA, 77:4216), and dp12.CHO cells (U.S. Pat. No. 5,721,121);monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCCCRL-1651); human embryonic kidney cells (e.g., 293 cells, or 293T cells,or 293 cells subcloned for growth in suspension culture, Graham et al.,1977, J. Gen. Virol., 36:59, or GnTI KO HEK293S cells, Reeves et al.,2002, PNAS, 99: 13419); baby hamster kidney cells (BHK, ATCC CCL-10);monkey kidney cells (CV1, ATCC CCL-70); African green monkey kidneycells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse sertoli cells(TM4, Mather, 1980, Biol. Reprod., 23:243-251); human cervical carcinomacells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); humanlung cells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065);mouse mammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat livercells (BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals NYAcad.Sci., 383:44-68); MCR 5 cells; FS4 cells. According to a particularembodiment, the cells are mammalian cells selected from Hek293 cells orCOS cells.

Exemplary non-mammalian cell lines include, but are not limited to, Sf9cells, baculovirus-insect cell systems (e.g., review Jarvis, VirologyVolume 310, Issue 1, May 25, 2003, Pages 1-7), plant cells such astobacco cells, tomato cells, maize cells, algae cells, or yeasts such asSaccharomyces species, Schizosaccharomyces species, Hansenula species,Yarrowia species or Pichia species. According to particular embodiments,the eukaryotic cells are yeast cells from a Saccharomyces species (e.g.,Saccharomyces cerevisiae), Schizosaccharomyces sp. (for example,Schizosaccharomyces pombe), a Hansenula species (e.g., Hansenulapolymorpha), a Yarrowia species (e.g., Yarrowia lipolytica), aKluyveromyces species (e.g., Kluyveromyces lactis), a Pichia species(e.g., Pichia pastoris), or a Komagalaella species (e.g., Komagataellapastoris). According to a specific embodiment, the eukaryotic cells arePichia cells, and in a most particular embodiment Pichia pastoris cells.

By “membrane protein” is understood a protein that is attached to orassociated with a membrane of a cell or an organelle. They are oftensubdivided into several categories including integral membrane proteins,peripheral membrane proteins and lipid-anchored proteins. Preferably,the membrane protein is an integral membrane protein that is permanentlybound to the lipid bilayer and which requires a detergent or anotherapolar solvent to be removed. Integral membrane proteins includetransmembrane proteins that are permanently attached to the lipidmembrane and span across the membrane one or several times. Examples ofsuitable membrane proteins include receptors such as GPCRs and growthfactor receptors; transmembrane ion channels such as ligand-gated andvoltage gated ion channels; transmembrane transporters such asneurotransmitter transporters; enzymes; carrier proteins; ion pumps;viral membrane proteins and supramolecular complexes thereof, amongstothers.

Depending on their intended use, membrane proteins as referred to hereincan be of any species, such as fungus (including yeast), nematode,virus, insect, plant, bird (e.g., chicken, turkey), reptile, or mammal(e.g., a mouse, rat, rabbit, hamster, gerbil, dog, cat, goat, pig, cow,horse, whale, monkey, or human).

In a specific embodiment, the membrane protein is a G-protein coupledreceptor or GPCR. GPCRs can be grouped on the basis of sequence homologyinto several distinct families. Although all GPCRs have a similararchitecture of seven membrane-spanning α-helices, the differentfamilies within this receptor class show no sequence homology to oneanother, thus suggesting that the similarity of their transmembranedomain structure might define common functional requirements. GPCRstructure and classification is generally well known in the art andfurther discussions of GPCRs may be found in Probst et al., 1992, DNACell Biol. 1:1; Marchese et al., 1994, Genomics 23:609; Rosenbaum etal., 2009, Nature 459:356; and the following books: Jurgen Wess (Ed)Structure-Function Analysis of G Protein-Coupled Receptors published byWiley-Liss (1st edition; Oct. 15, 1999); Kevin R. Lynch (Ed)Identification and Expression of G Protein-Coupled Receptors publishedby John Wiley & Sons (March 1998) and Tatsuya Haga (Ed), GProtein-Coupled Receptors, published by CRC Press (Sep. 24, 1999); andSteve Watson (Ed) G-Protein Linked Receptor Factsbook, published byAcademic Press (1st edition; 1994). A comprehensive view of the GPCRrepertoire was possible when the first draft of the human genome becameavailable. Fredriksson and colleagues divided 802 human GPCRs intofamilies on the basis of phylogenetic criteria. This showed that most ofthe human GPCRs can be found in five main families, termed Glutamate,Rhodopsin, Adhesion, Frizzled/Taste2 and Secretin (Fredriksson et al.,2003, Molecular Pharmacology 63:1256).

GPCRs include, without limitation, serotonin and olfactory receptors,glycoprotein hormone receptors, chemokine receptors, adenosinereceptors, biogenic amine receptors, melanocortin receptors,neuropeptide receptors, chemotactic receptors, somatostatin receptors,opioid receptors, melatonin receptors, calcitonin receptors, PTH/PTHrPreceptors, glucagon receptors, secretin receptors, latrotoxin receptors,metabotropic glutamate receptors, calcium receptors, GABA-B receptors,pheromone receptors, histamine receptors, protease-activated receptors,rhodopsins and other G-protein coupled seven transmembrane segmentreceptors. GPCRs also include these GPCR receptors associated with eachother as homomeric or heteromeric dimers or as higher-order oligomers.The amino acid sequences (and the nucleotide sequences of the cDNAswhich encode them) of GPCRs are readily available, for example, byreference to GenBank (on the World Wide Web at ncbi.nlm.nih.gov/entrez).Specific examples of GPCRs envisaged for increased production using thetools and methods provided herein include, but are not limited to,CXCR4, GPR3, rhodopsin, vasopressin receptor, β1 adrenergic receptor, β2adrenergic receptor, β3 adrenergic receptor, α1 adrenergic receptors andthe α2 adrenergic receptor, M1 muscarinic receptor, M2 muscarinicreceptor, M3 muscarinic receptor, M4 muscarinic receptor, M5 muscarinicreceptor, angiotensin II receptors.

Notably, fragments or portions, or mutants, variants, or analogues ofany of the aforementioned proteins and polypeptides are also includedamong the suitable proteins, polypeptides and peptides that can beproduced by the cells and methods presented herein. In particular, amembrane protein, as used herein, may be any naturally occurring ornon-naturally occurring (i.e., altered by man) membrane protein. Withinthis context, the term “naturally-occurring” means a membrane proteinthat is naturally produced (for example, and without limitation, by amammal, more specifically by a human, or by a virus, or by a plant, orby an insect, amongst others). Such membrane proteins are found innature. Analogously, the term “non-naturally occurring” means a membraneprotein that is not naturally produced. Wild-type membrane proteins thathave been made constitutively active through mutation, and variants ofnaturally occurring membrane proteins are examples of non-naturallyoccurring membrane proteins. Non-naturally occurring membrane proteinsmay have an amino acid sequence that is at least 80% identical to, atleast 90% identical to, at least 95% identical to or at least 99%identical to, a naturally-occurring membrane protein. Taking the CXCR4receptor as a particular non-limiting example of a GPCR within the scopeof the disclosure, it should be clear from the above that in addition tothe human CXCR4 receptor (e.g., the sequence described by Genbankaccession number EAX11616, Gene ID 7852), the mouse CXCR4 receptor(e.g., as described by Genbank accession number NP_034041, Gene ID12767) or other mammalian CXCR4 receptor may also be employed. Inaddition, the term is intended to encompass wild-type polymorphicvariants and certain other active variants from a particular species.For example, a “human CXCR4 receptor” has an amino acid sequence that isat least 95% identical to (e.g., at least 95% or at least 98% identicalto) the naturally occurring “human CXCR4” of Genbank accession numberEAX11616, Gene ID 7852. Further, it will be appreciated that thedisclosure also envisages membrane proteins, in particular GPCRs, with aloop deletion, or an N- and/or C-terminal deletion, or a substitution,or an insertion or addition in relation to its amino acid or nucleotidesequence, or any combination thereof (as defined hereinbefore), or amembrane protein in complex with another chemical entity such as one ormore interacting proteins or an agonist/antagonist/inverse agonist.

Thus, according to specific embodiments, the tools and methods formembrane protein expression provided herein can be further combined withknown improvements for membrane protein expression that typicallyinvolve production of variants. Examples thereof include, but are notlimited to, the use of a signal sequence specific to the species ofeukaryotic cell used rather than the membrane protein-specific signalsequence, the use of a truncated membrane protein (e.g., C-truncated)versus the use of an intact protein, the use of a membrane protein witha sequence insertion (e.g., a T4 lysozyme coding sequence in the 3^(rd)intracellular loop of a GPCR), and so on. Of course, these differentvariations can further be combined with each other.

According to still other specific embodiments, more than one, i.e., twoor more different proteins may be produced simultaneously. Preferably,at least one of the proteins is a membrane protein. The proteins may allbe membrane-bound, all be secreted proteins or a mixture thereof. Whenmore than one protein is produced, care will be taken that they can berecovered easily either separately or together. In a specificembodiment, even higher production is achieved by expressing multiplecopies of the protein to be expressed, e.g., as a polyprotein.

Preferably, the produced (membrane) proteins are functional, forinstance, the produced receptors remain capable of ligand binding and/orsignal transduction.

The host cell, according to the disclosure, is engineered so to expressa membrane protein as well as a binding domain directed against themembrane protein. By “binding domain” is meant any non-naturallyoccurring molecule or part thereof (as defined hereinbefore) that isdirected against a target membrane protein. In a preferred embodiment,the binding domains, as described herein, are protein scaffolds. Proteinscaffolds refer generally to folding units that form structures,particularly protein or peptide structures, that comprise frameworks forthe binding of another molecule, for instance, a protein (see, e.g.,review of Skerra, J. 2000, Molecular Recognition, 13:167). Accordingly,a binding domain can be derived from a naturally occurring molecule,e.g., from components of the innate or adaptive immune system, or it canbe entirely artificially designed. A binding domain can beimmunoglobulin-based or it can be based on domains present in proteinsincluding, but not limited to, microbial proteins, protease inhibitors,toxins, fibronectin, lipocalins, single chain antiparallel coiled coilproteins or repeat motif proteins. Examples of binding domains, whichare known in the art include, but are not limited to: antibodies, heavychain antibodies (hcAb), single domain antibodies (sdAb), minibodies,the variable domain derived from camelid heavy chain antibodies (VHH ornanobodies), the variable domain of the new antigen receptors derivedfrom shark antibodies (VNAR), alphabodies, protein A, protein G,designed ankyrin-repeat domains (DARPins), fibronectin type III repeats,anticalins, knottins, engineered CH2 domains (nanoantibodies), peptidesand proteins, lipopeptides (e.g., pepducins), DNA, and RNA (see, e.g.,Gebauer & Skerra, 2009, Curr Opin Chem Biol. 13:245; Skerra, 2000,Molecular Recognition, 13:167; Starovasnik et al., 1997, Proc Natl AcadSci USA. 94:10080; Binz et al., 2004, Nature Biotech., 22: 575; Koide etal., 1998, J. Mol. Biol., 284:1141; Dimitrov, 2009, MAbs. 1:26; Nygrenet al., 2008, FEBS J. 275:2668; WO2010066740). Frequently, whengenerating a particular type of binding domain using selection methods,combinatorial libraries comprising a consensus or framework sequencecontaining randomized potential interaction residues are used to screenfor binding to a molecule of interest, such as a protein.

The binding domain, according to the disclosure, may generally bedirected against any desired membrane protein, as describedhereinbefore, and may in particular be directed against anyconformational epitope of any membrane protein, preferably a functionalconformational state of any membrane protein (active, inactive, etc.).More particularly, the conformational epitope can be part of anintracellular or extracellular region, or an intramembraneous region, ora domain or loop structure of any desired membrane protein. According toparticular embodiments, the binding domains may be directed against anysuitable extracellular region, domain, loop or other extracellularconformational epitope of a membrane protein, but is preferably directedagainst one of the extracellular parts of the transmembrane domains ormore preferably against one of the extracellular loops that link thetransmembrane domains. Alternatively, the protein binding domains may bedirected against any suitable intracellular region, domain, loop orother intracellular conformational epitope of a membrane protein, but ispreferably directed against one of the intracellular parts of thetransmembrane domains or more preferably against one of theintracellular loops that link the transmembrane domains. A bindingdomain that specifically binds to a “three-dimensional” epitope or“conformational” epitope specifically binds to a tertiary (i.e.,three-dimensional) structure of a folded protein, and binds at muchreduced (i.e., by a factor of at least 2, 5, 10, 50 or 100) affinity tothe linear (i.e., unfolded, denatured) form of the protein.

According to other preferred embodiments, the expression of the proteinsin the host cell, as described herein, is preferably regulated byappropriate promoters. The choice of a promoter will typically depend onthe nature of the host cell. The choice further depends on the desiredtemporal expression of a particular protein, as described herein. Theproteins may be expressed constitutively or in an inducible way.Accordingly, the promoter may be a constitutive or inducible promoter.The conditions for inducing a promoter may be chosen from the followinggroup of inducing conditions: metabolic, or stress, or pH, ortemperature, or drug inducing conditions, or other. Promoters may bederived directly from naturally occurring genes, or may be synthesizedto combine regulatory sequences from different promoter regions.Preferably, the promoter is an exogenous promoter that will typically bestrong enough to ensure overexpression of the protein(s).

In a preferred embodiment, the promoter is an inducible promoter.Examples of inducible promoters useful for the practice of thedisclosure, in particular for yeast cells, include, without limitation,the promoter of a gene selected from the group comprising alcoholoxidase I (AOX1) (Tschopp et al., 1987, Nucleic Acids Res. 15:3859),alcohol oxidase II (AOXII) (Ohi et al., 1994, Mol. Gen. Genet. 243:489),formaldehyde dehydrogenase (FLD) (U.S. Pat. No. 6,730,499; Shen et al.,1998, Gene 216:93), galactokinase (GAL1) (Flick and Johnston 1990, Mol.Cell Biol. 10:4757), methanol oxidase (MOX) (GOdeke et al., 1994, Gene139:35), formate dehydrogenase (FMD) (Eggeling and Sahm 1978, Eur. J.Appl. Microbiol. Biotechnol. 5:197), mitochondrial alternative oxidase(AOD1) (Kern et al., 2007, Microbiology. 153:1250), peroxisomal acylcoenzyme A oxidase (POX1) (Koller et al., 1999, Yeast 15, 1035). Otherexamples typically for mammalian cells include the doxycycline-induciblesystem with reverse tetracycline-controlled transactivator (rtTA) andtetracycline-responsive element promoter (TRE) (Qin et al., 2010, PlosOne 5:e10611).

Alternatively, constitutive promoters may be used and include, forexample, the glyceraldehyde-3-phosphate dehydrogenase promoter (GAP)(Zhang et al., 2009, Mol. Biol. Rep 0.36: 1611) for expression in yeast;the simian virus 40 early promoter (SV40), the cytomegalovirusimmediate-early promoter (CMV), the human Ubiquitin C promoter (UBC),the human elongation factor 1a promoter (EF1A), the mousephosphoglycerate kinase 1 promoter (PGK), the chicken b-Actin promotercoupled with CMV early enhancer (CAGG) for expression in mammalian cells(Qin et al., 2010, Plos One 5:e10611).

According to a preferred embodiment, the disclosure relates to a hostcell engineered, as described hereinbefore, wherein the membrane proteinand the binding domain are co-expressed. By “co-expressed” is meanttemporally expressed at the same time or simultaneously expressed, whichcan be regulated by choosing the appropriate promoter(s) (as describedhereinbefore).

Further, it is preferred that upon expression in the host cell, themembrane protein and the binding domain are co-localized in a particularcellular compartment, such as in the ER, in the Golgi apparatus, at thecellular surface (meaning the membrane protein attached to the cellmembrane and the binding domain co-localized at the extracellular orintracellular side of the cell membrane). By “co-localized” is meantspatially expressed at the same cellular location. This can be done byoperably linking the membrane protein and/or the binding domain to anappropriate subcellular targeting sequence, as defined herein, such asan ER or Golgi localization signal, or a secretion signal, orcombinations thereof.

Depending on the application (see further herein), the membrane proteinand binding domain are preferably co-expressed and translocated to thecellular surface or to the extracellular space. This will typically beachieved by making use of secretion signals, so that the co-expressedproteins are transported through the secretory pathway: from the ER tothe cis-, medial- and trans-Golgi compartments, finally resulting insecretion into the extracellular medium, or otherwise integration in thecellular membrane, which is typically the case for membrane proteins.Preferably, both the membrane protein and the binding domain areoperably linked to a secretion signal. Even more preferably, both themembrane protein and the binding domain are operably linked to the samesecretion signal. The nature of the secretion signal will typically notdepend on the protein to be secreted, but on the type of eukaryoticcells used. As long as the secretion signal is functional in the celltype in which it is used (i.e., it results in secretion to theextracellular environment (or to the cellular membrane) of the proteinor peptide to which it is fused), this feature is not critical to thedisclosure. Thus, secretion signals from other organisms may be used, aslong as these signals lead to secretion in the eukaryotic cells used.Secretion signals are well known in the art and may be derivedfrom—typically the N-terminus of—proteins that are secreted, or may bemade synthetically (e.g., Tan et al., Protein engineering 2002, 15:337).Alternatively, they can be derived from genomic sequences usingcomputational methods (Klee et al., BMC Bioinformatics 2005, 6:256).Also, viral or bacterial or even hybrid secretion signals can be used.Further examples of signal peptides that can be used are described inWO2002/048187 (eukaryotic cells), Schaaf et al. (BMC Biotechnol. 2005;5: 30) (moss cells), EP549062. Specific secretion signals used in yeastinclude, e.g., α-factor secretory peptide, the PH05 secretory peptide,and the BARI secretion signal. In particular, secretion signals derivedfrom the Pichia genome sequence have been described in WO2010/135678.

Alternatively, the membrane protein and binding domain may also beco-expressed and retained in the ER or Golgi compartment. ER and Golgilocalization signals are well known in the art and may be derived fromproteins that are normally localized in the ER or Golgi for theirfunction. Again, localization sequences from one organism may functionin other organisms. For example, the membrane spanning region ofα-2,6-sialyltransferase from rats, an enzyme known to localize in therat trans Golgi, was shown to also localize a reporter gene (invertase)in the yeast Golgi (Schwientek, et al., 1995, J. Biol. Chem. 270:5483).Schwientek and co-workers have also shown that fusing 28 amino acids ofa yeast mannosyltransferase (Mnt1), a region containing an N-terminalcytoplasmic tail, a transmembrane region and eight amino acids of thestem region, to the catalytic domain of human GalT are sufficient forGolgi localization of an active GalT (Schwientek et al., 1995 J. Biol.Chem. 270 (10): 5483-5489). Other well-documented motifs are the KDELand HDEL motif for retention in the ER as well as other sequences listedin Table 5 of WO02/000879 such as the leader sequences from MnsI for ERlocalization, and leader sequences from Och1 and Mnt1 (Golgi-cislocalization), from Mnn2 (Golgi medial localization), from Mnn1 (Golgitrans localization), from alpha-2,6-sialyltransferase (trans-Golginetwork) and from beta-1,4-galactosyltransferase I (Golgi localization).

Alternatively, the membrane protein and binding domain may also beco-expressed and deposited in inclusion bodies in the cell, or inmembrane-bound organelles or in structures with similar functions. Whencells are part of an organism that is used for production (e.g., a plantinstead of a plant cell culture), the co-expressed proteins may beproduced in or transported to specific organs or tissues of the organismfrom which it can be recovered (e.g., glands or trichomes). It should benoted that, particularly in cases where the protein is not secreted, itis possible that the protein is deposited in an inactive form. Thus,additional refolding or re-activating steps may be needed in order toobtain a physiologically relevant form of the protein.

According to a preferred embodiment of the disclosure, it isparticularly envisaged that the binding domain which is co-expressedwith the membrane protein, as described hereinbefore, is derived from aninnate or adaptive immune system. Preferably, the binding domain isderived from an immunoglobulin. Preferably, the protein binding domain,according to the disclosure, is an antibody or a derivative thereof. Theterm “antibody” (Ab) refers generally to a polypeptide encoded by animmunoglobulin gene, or functional fragments thereof that specificallybinds and recognizes an antigen, and is known to the person skilled inthe art. A conventional immunoglobulin (antibody) structural unitcomprises a tetramer. Each tetramer is composed of two identical pairsof polypeptide chains, each pair having one “light” (about 25 kDa) andone “heavy” chain (about 50-70 kDa). The N-terminus of each chaindefines a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The terms variable lightchain (VL) and variable heavy chain (VH) refer to these light and heavychains, respectively. The term “antibody” is meant to include wholeantibodies, including single-chain whole antibodies, and antigen-bindingfragments. In some embodiments, antigen-binding fragments may beantigen-binding antibody fragments that include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (dsFv) and fragments comprising orconsisting of either a VL or VH domain, and any combination of those orany other functional portion of an immunoglobulin peptide capable ofbinding to the target antigen. The term “antibodies” is also meant toinclude heavy chain antibodies, or functional fragments thereof, such assingle domain antibodies, more specifically, VHHs or nanobodies, asdefined further herein.

Preferably, the binding domain is an immunoglobulin single variabledomain. More preferably, the binding domain comprises an amino acidsequence comprising 4 framework regions and 3complementarity-determining regions, preferably in a sequenceFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, or any suitable fragment thereof (whichwill then usually contain at least some of the amino acid residues thatform at least one of the complementarity-determining regions). Bindingdomains comprising 4 FRs and 3 CDRs are known to the person skilled inthe art and have been described, as a non-limiting example, inWesolowski et al., 2009, Med. Microbiol. Immunol. 198:157.

Preferably, the binding domain, according to the disclosure, is derivedfrom a camelid antibody. More preferably, the binding domain, accordingto the disclosure, comprises an amino acid sequence of a nanobody, orany suitable fragment thereof. More specifically, the protein bindingdomain is a nanobody or any suitable fragment thereof. A “nanobody”(Nb), as used herein, refers to the smallest antigen binding fragment orsingle variable domain (“VHH”) derived from a naturally occurring heavychain antibody and is known to the person skilled in the art. They arederived from heavy chain only antibodies, seen in camelids(Hamers-Casterman et al., 1993, Nature 363:446; Desmyter et al., 1996,Nat. Struct. Biol. 3:803). In the family of “camelids” immunoglobulinsdevoid of light polypeptide chains are found. “Camelids” comprise oldworld camelids (Camelus bactrianus and Camelus dromedarius) and newworld camelids (for example, Lama paccos, Lama glama, Lama guanicoe andLama vicugna). The single variable domain heavy chain antibody is hereindesignated as a nanobody or a VHH. NANOBODY®, NANOBODIES® and NANOCLONE®are trademarks of Ablynx NV (Belgium). The small size and uniquebiophysical properties of Nbs excel conventional antibody fragments forthe recognition of uncommon or hidden epitopes and for binding intocavities or active sites of protein targets. Further, Nbs can bedesigned as bispecific and bivalent antibodies or attached to reportermolecules (Conrath et al., 2001, Antimicrob. Agents Chemother. 45:2807). Nbs are stable and rigid single domain proteins that can easilybe manufactured and survive the gastro-intestinal system. Therefore, Nbscan be used in many applications including drug discovery and therapy(Saerens et al., 2008, Curr. Opin. Pharmacol. 8:600) but also as aversatile and valuable tool for purification, functional study andcrystallization of proteins (Conrath et al., 2009, Protein Sci. 18:619).In that regard, a particular class of nanobodies that act ascrystallization chaperones binding conformational epitopes of nativetargets are called Xaperones and are also envisaged here. Xaperones areunique tools in structural biology. XAPERONE™ is a trademark of VIB andVUB (Belgium). Major advantages for the use of camelid antibodyfragments as crystallization aid are that Xaperones (1) bind crypticepitopes and lock proteins in unique native conformations, (2) increasethe stability of soluble proteins and solubilized membrane proteins, (3)reduce the conformational complexity of soluble proteins and solubilizedmembrane proteins, (4) increase the polar surface enabling the growth ofdiffracting crystals, (5) sequester aggregative or polymerizingsurfaces, (6) allow to affinity-trap active protein.

The nanobodies, according to the disclosure, generally comprise a singleamino acid chain that can be considered to comprise 4 “frameworksequences” or FR's and 3 “complementarity-determining regions” or CDR's(as defined hereinbefore). Non-limiting examples of nanobodies of thedisclosure are described in more detail further herein. It should beclear that framework regions of nanobodies may also contribute to thebinding of their antigens (Desmyter et al., 2002, J. Biol. Chem.277:23645; Korotkov et al., 2009, Structure 17:255).

It should be noted that the term nanobody, as used herein, in itsbroadest sense is not limited to a specific biological source or to aspecific method of preparation. For example, the nanobodies of thedisclosure can generally be obtained: (1) by isolating the VHH domain ofa naturally occurring heavy chain antibody; (2) by expression of anucleotide sequence encoding a naturally occurring VHH domain; (3) by“humanization” of a naturally occurring VHH domain or by expression of anucleic acid encoding a such humanized VHH domain; (4) by “camelization”of a naturally occurring VH domain from any animal species, and inparticular from a mammalian species, such as from a human being, or byexpression of a nucleic acid encoding such a camelized VH domain; (5) by“camelization” of a “domain antibody” or “Dab,” as described in the art,or by expression of a nucleic acid encoding such a camelized VH domain;(6) by using synthetic or semi-synthetic techniques for preparingproteins, polypeptides or other amino acid sequences known per se; (7)by preparing a nucleic acid encoding a nanobody using techniques fornucleic acid synthesis known per se, followed by expression of thenucleic acid thus obtained; and/or (8) by any combination of one or moreof the foregoing.

According to a preferred embodiment, the nanobodies or VHHs are directedagainst a functional conformational state of a membrane protein, asdescribed hereinbefore. Although naive or synthetic libraries ofnanobodies (for examples of such libraries, see WO9937681, WO0043507,WO0190190, WO03025020 and WO03035694) may contain conformational bindersagainst a membrane protein in a functional conformational state, apreferred embodiment of this disclosure includes the immunization of aCamelidae with a membrane protein in a functional conformational state,optionally bound to a ligand, to expose the immune system of the animalwith the conformational epitopes that are unique to the membrane proteinin that particular conformation (for example, agonist-bound GPCR so asto raise antibodies directed against a GPCR in its active conformationalstate; or antagonist-bound GPCR so as to raise antibodies directedagainst a GPCR in its inactive conformational state). Thus, as furtherdescribed herein, such VHH sequences can preferably be generated orobtained by suitably immunizing a species of Camelid with a targetmembrane protein, preferably a membrane protein in a functionalconformational state (i.e., so as to raise an immune response and/orheavy chain antibodies directed against the membrane protein), byobtaining a suitable biological sample from the Camelid (such as a bloodsample, or any sample of B-cells), and by generating VHH sequencesdirected against the membrane protein, starting from the sample. Suchtechniques will be clear to the skilled person. Yet another techniquefor obtaining the desired VHH sequences involves suitably immunizing atransgenic mammal that is capable of expressing heavy chain antibodies(i.e., so as to raise an immune response and/or heavy chain antibodiesdirected against a membrane protein in a functional conformationalstate), obtaining a suitable biological sample from the transgenicmammal (such as a blood sample, or any sample of B-cells), and thengenerating VHH sequences directed against the membrane protein startingfrom the sample, using any suitable technique known per se. For example,for this purpose, the heavy chain antibody-expressing mice and thefurther methods and techniques described in WO02085945 and in WO04049794can be used.

Non-limiting examples of the nanobodies, according to the disclosure,include, but are not limited to, nanobodies as defined by SEQ ID NOS:1-6(see Table 1). The delineation of the CDR sequences is based on the IMGTunique numbering system for V-domains and V-like domains (Lefranc etal., 2003, Developmental and Comparative Immunology 27:55). In aspecific embodiment, the above nanobodies can comprise at least one ofthe complementarity-determining regions (CDRs) with an amino acidsequence selected from SEQ ID NOS:7-24 (see Table 2). More specifically,the above nanobodies can be selected from the group comprising SEQ IDNOS: 1-6, or a functional fragment thereof. A “functional fragment” or a“suitable fragment,” as used herein, may, for example, comprise one ofthe CDR loops. Preferably, the functional fragment comprises CDR3. Morespecifically, the nanobodies consist of any of SEQ ID NOS:1-6 and thefunctional fragment of said nanobodies consist of any of SEQ IDNOS:7-24. Further, nucleic acid sequences encoding any of the abovenanobodies or functional fragments are also envisaged in the disclosure.

It is also within the scope of the disclosure to use natural orsynthetic analogs, mutants, variants, alleles, homologs and orthologs(herein collectively referred to as “analogs”) of the binding domains,according to the disclosure, preferably to the nanobodies, and inparticular analogs of the nanobodies of SEQ ID NOS:1-6 (see Table 1).Thus, according to one embodiment of the disclosure, the term “nanobodyof the disclosure” in its broadest sense also covers such analogs.Generally, in such analogs, one or more amino acid residues may havebeen replaced, deleted and/or added, compared to the nanobodies of thedisclosure, as defined herein. Such substitutions, insertions, deletionsor additions may be made in one or more of the framework regions and/orin one or more of the CDR's. Analogs, as used herein, are sequenceswherein each or any framework region and each or anycomplementarity-determining region shows at least 80% identity,preferably at least 85% identity, more preferably 90% identity, evenmore preferably 95% identity with the corresponding region in thereference sequence (i.e., FR1_analog versus FR1_reference, CDR1_analogversus CDR1_reference, FR2_analog versus FR2_reference, CDR2_analogversus CDR2_reference, FR3_analog versus FR3_reference, CDR3_analogversus CDR3_reference, FR4_analog versus FR4_reference), as measured ina BLASTp alignment (Altschul et al., 1997, Nucleic Acids Res. 25:3389;FR and CDR definitions according to IMGT unique numbering system forV-domains and V-like domains (Lefranc et al., 2003, Developmental andComparative Immunology 27:55)). Non-limiting examples include analogs ofthe CDR's of the nanobodies of SEQ ID NOS:1-6, the CDR's correspondingwith SEQ ID NOS:7-24 (see Table 2).

By means of non-limiting examples, a substitution may, for example, be aconservative substitution, as defined herein, and/or an amino acidresidue may be replaced by another amino acid residue that naturallyoccurs at the same position in another VHH domain. Thus, any one or moresubstitutions, deletions or insertions, or any combination thereof, thateither improve the properties of the nanobody of the disclosure or thatat least do not detract too much from the desired properties or from thebalance or combination of desired properties of the nanobody of thedisclosure (i.e., to the extent that the nanobody is no longer suitedfor its intended use) are included within the scope of the disclosure. Askilled person will generally be able to determine and select suitablesubstitutions, deletions, insertions, additions, or suitablecombinations of thereof, based on the disclosure herein and optionallyafter a limited degree of routine experimentation, which may, forexample, involve introducing a limited number of possible substitutionsand determining their influence on the properties of the nanobodies thusobtained.

For example, and depending on the host organism used to express thebinding domain of the disclosure, preferably the nanobody, suchdeletions and/or substitutions may be designed in such a way that one ormore sites for post-translational modification (such as one or moreglycosylation sites) are removed, as will be within the ability of theperson skilled in the art. Alternatively, substitutions or insertionsmay be designed so as to introduce one or more sites for attachment offunctional groups, residues or moieties. Examples of modifications, aswell as examples of amino acid residues within the binding domainsequence, preferably the nanobody sequence, that can be modified (i.e.,either on the protein backbone but preferably on a side chain), methodsand techniques that can be used to introduce such modifications and thepotential uses and advantages of such modifications will be clear to theskilled person.

A particular type of modification may comprise the introduction of oneor more detectable labels or other signal-generating groups or moieties,depending on the intended use of the labeled binding domain, inparticular the nanobody. Suitable labels and techniques for attaching,using and detecting them will be clear to the skilled person, and, forexample, include, but are not limited to, fluorescent labels,phosphorescent labels, chemiluminescent labels or bioluminescent labels,radio-isotopes, metals, metals chelates or metallic cations or othermetals or metallic cations that are particularly suited for in vivo, invitro or in situ diagnosis and imaging (including immunoassays known perse such as ELISA, RIA, EIA and other “sandwich assays,” etc.), as wellas chromophores and enzymes. Other suitable labels will be clear to theskilled person, and, for example, include moieties that can be detectedusing NMR or ESR spectroscopy. Yet another modification may comprise theintroduction of a functional group that is one part of a specificbinding pair, such as the biotin-(strept)avidin binding pair.

In a particular embodiment, the nanobody of the disclosure is bivalentand formed by bonding, chemically or by recombinant DNA techniques,together two monovalent single domain of heavy chains. In anotherparticular embodiment, the nanobody of the disclosure is bi-specific andformed by bonding together two variable domains of heavy chains, eachwith a different specificity. Similarly, polypeptides comprisingmultivalent or multi-specific nanobodies are included here asnon-limiting examples. Preferably, a monovalent nanobody of thedisclosure is such that it will bind to an extracellular part, region,domain, loop or other extracellular epitope of a functionalconformational state of a membrane protein, with a dissociation constantof less than 500 nM, preferably less than 200 nM, more preferably lessthan 10 nM, such as less than 500 pM. Alternatively, a monovalentnanobody of the disclosure is such that it will bind to an intracellularpart, region, domain, loop or other intracellular epitope of afunctional conformational state of a membrane protein, with andissociation constant of less than 500 nM, preferably less than 200 nM,more preferably less than 10 nM, such as less than 500 pM. Also,according to this aspect, any multivalent or multispecific, as definedherein, nanobody of the disclosure may also be suitably directed againsttwo or more different extracellular or intracellular parts, regions,domains, loops or other extracellular or intracellular epitopes on thesame antigen, for example, against two different extracellular orintracellular loops or against two different extracellular orintracellular parts of the transmembrane domains. Such multivalent ormultispecific nanobodies of the disclosure may also have (or beengineered and/or selected for) increased avidity and/or improvedselectivity for the desired target protein, and/or for any other desiredproperty or combination of desired properties that may be obtained bythe use of such multivalent or multispecific nanobodies. In a particularembodiment, such multivalent or multispecific nanobodies of thedisclosure may also have (or be engineered and/or selected for) improvedefficacy in modulating signaling activity of a target protein.

Various methods may be used to determine specific binding between thebinding domain and a target membrane protein, including, for example,enzyme linked immunosorbent assays (ELISA), flow cytometry, surfaceplasmon resonance assays, and the like, which are common practice in theart, for example, in discussed in Sambrook et al., 2001. MolecularCloning, A Laboratory Manual. Third Edition. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. It will be appreciated thatfor this purpose often a unique label or tag will be used, such as apeptide label, a nucleic acid label, a chemical label, a fluorescentlabel, or a radio frequency tag.

It should be clear that membrane proteins, as used herein, areconformationally complex proteins that exhibit a spectrum functionalbehavior in response to natural and synthetic ligands. Thus, in apreferred embodiment, the binding domains, as described hereinbefore,are capable of stabilizing, or otherwise, increasing the stability of aparticular functional conformational state, as defined herein, of amembrane protein, such as an active or an inactive state, etc.Preferably, the binding domain is capable of inducing the formation of afunctional conformational state in a membrane protein upon binding theprotein. Taking a GPCR as a non-limiting example, the functionalconformation state can be a basal conformational state, an activeconformational state or an inactive conformational state. Preferably,the membrane protein is stabilized in a drugable conformation.

The wording “inducing” or “forcing” or “locking” or “trapping” or“fixing” or “freezing” or “stabilizing,” with respect to a functionalconformational state of a membrane protein, as used herein, refers tothe retaining or holding of a membrane protein in a subset of thepossible conformations that it could otherwise assume, due to theeffects of the interaction of the protein with the binding domain,according to the disclosure. Accordingly, a protein that is“conformationally trapped” or “conformationally fixed” or“conformationally locked” or “conformationally frozen,” or in a“stabilized conformation,” as used herein, is one that is held in asubset of the possible conformations that it could otherwise assume, dueto the effects of the interaction of the protein with the bindingdomain, according to the disclosure. Within this context, a bindingdomain that specifically or selectively binds to a specific conformationor conformational state of a protein refers to a binding domain thatbinds with a higher affinity to a protein in a subset of conformationsor conformational states than to other conformations or conformationalstates that the protein may assume. One of skill in the art willrecognize that binding domains that specifically or selectively bind toa specific conformation or conformational state of a protein willstabilize this specific conformation or conformational state.

It will be appreciated that having increased stability with respect tostructure and/or a particular biological activity of a membrane proteinmay also be a guide to the stability to other denaturants or denaturingconditions including heat, a detergent, a chaotropic agent and anextreme pH. Accordingly, in a further embodiment, the binding domain,according to the disclosure, is capable of increasing the stability of afunctional conformational state of a membrane protein undernon-physiological conditions induced by dilution, concentration, buffercomposition, heating, cooling, freezing, detergent, chaotropic agent,pH. In contrast to water-soluble proteins, thermodynamic studies ofmembrane protein folding and stability have proven to be extremelychallenging, and complicated by the difficulty of finding conditions forreversible folding. Unfolding of helical membrane proteins induced bymost methods, such as thermal and chemical approaches, is irreversibleas reviewed by Stanley and Fleming (2008, Archives of Biochemistry andBiophysics 469:46). The term “thermostabilize,” “thermostabilizing,”“increasing the thermostability of,” as used herein, therefore, refersto the functional rather than to the thermodynamic properties of amembrane protein and to the protein's resistance to irreversibledenaturation induced by thermal and/or chemical approaches including,but not limited to, heating, cooling, freezing, chemical denaturants,pH, detergents, salts, additives, proteases or temperature. Irreversibledenaturation leads to the irreversible unfolding of the functionalconformations of the protein, loss of biological activity andaggregation of the denaturated protein. The term “(thermo)stabilize,”“(thermo)stabilizing,” “increasing the (thermo)stability of,” as usedherein, applies to membrane proteins embedded in lipid particles orlipid layers (for example, lipid monolayers, lipid bilayers, and thelike) and to membrane proteins that have been solubilized in detergent.

Thus, in another preferred embodiment, the binding domain, according tothe disclosure, is capable of increasing the thermostability of afunctional conformational state of a membrane protein when co-expressedin a host cell. In relation to an increased stability to heat, this canbe readily determined by measuring ligand binding or by usingspectroscopic methods such as fluorescence, CD or light scattering thatare sensitive to unfolding at increasing temperatures. It is preferredthat the binding domain is capable of increasing the stability asmeasured by an increase in the thermal stability of a membrane proteinin a functional conformational state with at least 2° C., at least 5°C., at least 8° C., and more preferably at least 10° C. or 15° C. or 20°C. In relation to an increased stability to a detergent or to achaotrope, typically, the GPCR is incubated for a defined time in thepresence of a test detergent or a test chaotropic agent and thestability is determined using, for example, ligand binding or aspectroscopic method, optionally at increasing temperatures as discussedabove. According to still another preferred embodiment, the proteinbinding domain, according to the disclosure, is capable of increasingthe stability to extreme pH of a functional conformational state of amembrane protein. In relation to an extreme of pH, a typical test pHwould be chosen, for example, in the range 6 to 8, the range 5.5 to 8.5,the range 5 to 9, the range 4.5 to 9.5, more specifically in the range4.5 to 5.5 (low pH) or in the range 8.5 to 9.5 (high pH).

According to yet another specific embodiment, the host cell of thedisclosure is a glyco-engineered host cell. A “glyco-engineered hostcell” refers to a host cell that has been genetically modified so thatit expresses proteins with an altered N-glycan structure and/or O-glycanstructure as compared to in a wild-type background. Typically, thenaturally occurring modifications on glycoproteins have been altered bygenetic engineering of enzymes involved in the glycosylation pathway. Ingeneral, sugar chains in N-linked glycosylation may be divided in threetypes: high-mannose (typically yeast), complex (typically mammalian) andhybrid type glycosylation. Besides that, a variety of O-glycan patternsexist, for example, with yeast oligomannosylglycans differing frommucin-type O-glycosylation in mammalian cells. The different types of N-and O-glycosylation are all well known to the skilled person and definedin the literature. Considerable effort has been directed towards theidentification and optimization of strategies for the engineering ofeukaryotic host cells that produce glycoproteins having a desired N-and/or O-glycosylation pattern and are known in the art (e.g., De Pourcqet al., 2010, Appl. Microbiol. Biotechnol. 87:1617). One non-limitingexample of such a glyco-engineered expression system is described inpatent application PCT/EP2009/060348 and relates to a eukaryotic hostcell expressing both an endoglucosaminidase and a target protein, andwherein the expressed target proteins are characterized by a uniformN-glycosylation pattern (in particular one GlcNAc residue or amodification thereof such as GlcNAc modified with galactose or galactoseand sialic acid). This can be, for example, particularly advantageous incrystallization studies of glycoproteins. Also encompassed are hostcells genetically modified so that they express proteins orglycoproteins in which the glycosylation pattern is human-like orhumanized (i.e., complex-type glycoproteins). This can be achieved byproviding host cells, in particular lower eukaryotic host cells, havinginactivated endogenous glycosylation enzymes and/or comprising at leastone other exogenous nucleic acid sequence encoding at least one enzymeneeded for complex glycosylation. Endogenous glycosylation enzymes,which could be inactivated, include the alpha-1,6-mannosyltransferaseOch1p, Alg3p, alpha-1,3-mannosyltransferase of the Mnn1p family,beta-1,2-mannosyltransferases. Enzymes needed for complex glycosylationinclude, but are not limited to: N-acetylglucosaminyl transferase I,N-acetylglucosaminyl transferase II, mannosidase II,galactosyltransferase, fucosyltransferase and sialyltransferase, andenzymes that are involved in donor sugar nucleotide synthesis ortransport. Still other glyco-engineered host cells, in particular yeastcells, that are envisaged here are characterized in that at least oneenzyme involved in the production of high mannose structures (highmannose-type glycans) is not expressed. Enzymes involved in theproduction of high mannose structures typically aremannosyltransferases. In particular, alpha-1,6-mannosyltransferaseOch1p, Alg3p, alpha-1,3-mannosyltransferase of the Mnn1p family,beta-1,2-mannosyltransferases may not be expressed. Thus, a host cellcan additionally or alternatively be engineered to express one or moreenzymes or enzyme activities, which enable the production of particularN-glycan structures at a high yield. Such an enzyme can be targeted to ahost subcellular organelle in which the enzyme will have optimalactivity, for example, by means of signal peptide not normallyassociated with the enzyme. It should be clear that the enzymesdescribed herein and their activities are well-known in the art.

Still other preferred host cells are: eukaryotic host cells as describedin patent application PCT/EP2011/059959, that have been engineered so todisplay increased membrane formation from which increased levels ofproteins can be recovered; eukaryotic host cells overexpressing HAC1 asdescribed in Guerfal et al., 2010, Microbial. Cell Factories, 9:49.

According to particularly envisaged embodiments, cell cultures of hostcells of the disclosure are also provided, as well as membranepreparations derived thereof (including the target membrane proteinattached to either the cell surface membrane or retained in anothersubcellular membrane compartment). Membrane preparations includemembrane fragments as well as membrane-detergent extracts and can beprepared according to known techniques, for example, as reviewed indetail in Cooper, 2004, J. Mol. Recognit. 17:286, incorporated herein byreference. A membrane preparation is also meant to include any liposomalcomposition which may comprise natural or synthetic lipids or acombination thereof. Examples of membrane or liposomal compositionsinclude, but are not limited to, organelles, membrane preparations,Virus Like Lipoparticles, lipid layers (bilayers and monolayers), lipidvesicles, high-density lipoparticles (e.g., nanodisks), and the like.

In another aspect, the disclosure provides vector constructs comprisingan exogenous nucleic acid sequence, as described hereinbefore.Non-limiting examples are provided in the Example section.

Applications

In the disclosure, it was surprisingly found that co-expressing amembrane protein with a binding domain directed against the membraneprotein results in an increased expression of the heterologouslyexpressed membrane protein, which can be useful for many applications,as described further herein.

The host cells, or cell cultures or membrane preparations derivedthereof, can be used to produce higher amounts of often poorly expressedand unstable target membrane proteins, optionally stabilized in aparticular conformation, either at the cellular surface or in other cellcompartments. As such, they are of immediate use as research tool for awide range of functional and/or structural studies.

Accordingly, in a further aspect, the disclosure provides a method ofproducing or enhancing the production of a membrane protein in a hostcell comprising the steps of:

-   -   a. Providing a host cell, according to the disclosure, as        described hereinbefore,    -   b. Culturing the host cell under conditions suitable for        co-expressing the membrane protein and the binding domain        directed against the membrane protein.

In addition, also envisaged is a method of producing or enhancing theproduction of a membrane protein in a functional conformation in a hostcell, the method comprising the steps of:

-   -   a. Providing a host cell, according to the disclosure, as        described hereinbefore,    -   b. Culturing the host cell under conditions suitable for        co-expressing the membrane protein and the binding domain        directed against the membrane protein, in the presence of a        ligand, such as an agonist, an antagonist, an inverse agonist.

According to specific embodiments of the above methods, the engineeredhost cells of the disclosure are cultured in the presence of aconformation-selective ligand, as defined herein. More specifically, theengineered host cells of the disclosure are cultured in the presence ofan agonist, or an antagonist, or an inverse agonist, or a biasedagonist; and/or a positive allosteric modulator, or a negativeallosteric modulator, all as defined herein.

During or after the protein production in the host cells, the protein orproteins of interest can be recovered from the cells. Accordingly, themethods of protein production may, optionally, also comprising the stepof isolating the expressed protein, either alone, or in complex with thebinding domain and/or with a ligand and/or with one or more downstreaminteracting proteins. This typically involves recovery of the materialwherein the protein(s) are present (e.g., a cell lysate or specificfraction thereof, the medium wherein the protein is secreted) andsubsequent purification of the protein. Means that may be employed tothis end are known to the skilled person and include specificantibodies, tags fused to the proteins, affinity purification columns,and the like. Advantageously, the binding domain itself may be a usefultool to purify the membrane protein in a functional conformationalstate, by fusing the membrane protein, and binding domain-coding genesto a different tag, which can then be used as consecutive handles foraffinity chromatography. When using double affinity chromatography, onlymembrane proteins that interact specifically with the binding domain areisolated. Moreover, they are all forced in the same functionalconformation, for example, and without the purpose of being limitative,an active or inactive state of a GPCR, depending on the agonistic orantagonistic characteristics of the binding domain, respectively.

Capturing and/or purifying the membrane protein in a particularconformation, either alone or in complex with a stabilizing bindingdomain, will allow subsequent crystallization of the complex with lessengineering steps, resulting in crystals of the membrane protein in afunctional conformational state. These structures are considered to bemuch more true to life than those in which part of the protein ismutated, deleted or replaced by other stabilizing protein structures.For example, almost all currently unravelled GPCR structures are fusionproteins of the GPCR, where intracellular loop 3 is exchanged for T4lysozyme. This intervention can, however, cause conformational changesthat are naturally not present. Therefore, it is of particular advantageto isolate and crystallize a membrane protein in a relevant conformationstarting from a host cell culture co-expressing in high amounts themembrane protein and a stabilizing binding domain. All these advantageswill make structure determination of membrane proteins much easier inthe future.

In practice, methods and techniques for crystallography and structuredetermination are all well known by the skilled in the art. A variety ofspecialized crystallization methods for membrane proteins exist, andmany of these are reviewed in Caffrey, 2003, J Struct. Biol. 142:108. Ingeneral terms, the methods are lipid-based methods that include addinglipid to the membrane protein-binding domain complex prior tocrystallization. Many of these methods, including the lipidic cubicphase crystallization method and the bicelle crystallization method,exploit the spontaneous self-assembling properties of lipids anddetergent as vesicles (vesicle-fusion method), discoidal micellcs(bicelle method), and liquid crystals or mesophases (in meso orcubic-phase method). Lipidic cubic phases crystallization methods aredescribed in, for example: Landau et al., 1996, Proc. Natl. Acad. Sci.93:14532; Gouaux 1998, Structure 6:5; Rummel et al., 1998, J. Struct.Biol. 121:82; Nollert et al., 2004, Methods 34:348, which publicationsare incorporated by reference for disclosure of those methods. Bicellecrystallization methods are described in, for example: Faham et al.,2005, Protein Sci. 14:836; Faham et al., 2002, J MoI Biol. 316:1, whichpublications are incorporated by reference for disclosure of thosemethods.

It has been proven that protein binding domains, in particular VHHs ornanobodies, are very useful tools to improve the diffraction quality ofprotein crystals, in particular membrane protein crystals (e.g., GPCRs)so that the protein crystal structure can be solved (see, e.g.,Rasmussen et al., 2011, Nature 469:175). The structure of a protein, inparticular a membrane protein, includes the primary, secondary, tertiaryand, if applicable, quaternary structure of the protein. “Solving thestructure,” as used herein, refers to determining the arrangement ofatoms or the atomic coordinates of a protein, and is often done by abiophysical method, such as X-ray crystallography. The acquiredstructural information can then be used to help guide drug discovery.

Other applications are particularly envisaged by making direct use ofthe host cells or cell cultures, according to the disclosure, or byusing membrane preparations derived thereof, which will be describedfurther herein, including compound screening and immunizations.

In the process of compound screening, lead optimization and drugdiscovery, there is a requirement for faster, more effective, lessexpensive and especially information-rich screening assays that providesimultaneous information on various compound characteristics and theireffects on various cellular pathways (i.e., efficacy, specificity,toxicity and drug metabolism). Thus, there is a need to quickly andinexpensively screen large numbers of compounds in order to identify newspecific ligands of a protein of interest, preferablyconformation-selective ligands, which may be potential new drugcandidates. The disclosure solves this problem by providing host cellsco-expressing at the cellular surface or in a particular cellularmembrane fraction high levels of membrane proteins and binding domainsdirected against these membrane proteins. Preferably, the bindingdomains are conformation-selective binding domains that allostericallystabilize and/or lock the membrane protein in a functionalconformational state, for example, Rasmussen et al., 2011, Nature469:175. Such host cells, as well as host cell cultures or membranepreparations derived thereof, can then be used as immunogens orselection reagents for screening in a variety of contexts. Taking a GPCRas an example, a major advantage of the combined features of highexpression levels and conformational stabilization by the binding domainis that the GPCR can be kept in a stabilized drugable conformation, forexample, the active state conformation. This will allow to quickly andreliably screen for and differentiate between receptor agonists, inverseagonists, antagonists and/or modulators as well as inhibitors of GPCRsand GPCR-dependent pathways, so increasing the likelihood of identifyinga ligand with the desired pharmacological properties. Even morepreferably, the binding domains increase the thermostability of themembrane protein in a particular functional conformational state, thusprotecting the membrane protein irreversible or thermal denaturationinduced by the non-native conditions used in compound screening and drugdiscovery, without the need to rely on, for example, mutant GPCRs withincreased stability. As such, screening performance for diseaseindications, associated with a particular functional conformer of atarget membrane protein will be improved by making use of the host cellsof the disclosure.

Thus, according to a preferred embodiment, the disclosure encompassesthe use of the host cells, or host cell cultures, or membranepreparations derived thereof, according to the disclosure and asdescribed hereinbefore, in screening and/or identification programs forconformation-selective binding partners of a membrane protein, whichultimately might lead to potential new drug candidates.

According to one embodiment, the disclosure envisages a method ofidentifying compounds capable of selectively binding to a functionalconformational state of a membrane protein, the method comprising thesteps of:

-   -   (i) Providing a host cell or host cell culture or membrane        preparation derived thereof, according to the disclosure,        harboring a membrane protein in a functional conformational        state, and    -   (ii) Providing a test compound, and    -   (iii) Evaluating whether the test compound binds to the        functional conformational state of the membrane protein, and    -   (iv) Selecting a compound that selectively binds to the        functional conformational state of the membrane protein.

Specific preferences for the host cells, cultures and membranepreparations thereof are as defined above with respect to the firstaspect of the disclosure.

Screening assays for drug discovery can be solid phase (e.g., beads,columns, slides, chips or plates) or solution phase assays, e.g., abinding assay, such as radioligand binding assays. In high-throughputassays, it is possible to screen up to several thousand differentcompounds in a single day in 96-, 384- or 1536-well formats. Forexample, each well of a microtiter plate can be used to run a separateassay against a selected potential modulator, or, if concentration orincubation time effects are to be observed, every 5-10 wells can test asingle modulator. Thus, a single standard microtiter plate can assayabout 96 modulators. It is possible to assay many plates per day; assayscreens for up to about 6.000, 20.000, 50.000 or more differentcompounds are possible today. Preferably, a screening for membraneprotein conformation-specific compounds will be performed starting fromhost cells, or host cell cultures, or membrane preparations derivedthereof.

Various methods may be used to determine binding between the stabilizedmembrane protein and a test compound, including, for example, enzymelinked immunosorbent assays (ELISA), surface Plasmon resonance assays,chip-based assays, immunocytofluorescence, yeast two-hybrid technologyand phage display, which are common practice in the art, for example, inSambrook et al., 2001, Molecular Cloning, A Laboratory Manual. ThirdEdition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.Other methods of detecting binding between a test compound and amembrane protein include ultrafiltration with ion spray massspectroscopy/HPLC methods or other (bio)physical and analytical methods.Fluorescence Energy Resonance Transfer (FRET) methods, for example, wellknown to those skilled in the art, may also be used. It will beappreciated that a bound test compound can be detected using a uniquelabel or tag associated with the compound, such as a peptide label, anucleic acid label, a chemical label, a fluorescent label, or a radiofrequency tag, as described further herein.

The compounds to be tested can be any small chemical compound, or amacromolecule, such as a protein, a sugar, nucleic acid or lipid.Typically, test compounds will be small chemical compounds, peptides,antibodies or fragments thereof. It will be appreciated that in someinstances the test compound may be a library of test compounds. Inparticular, high-throughput screening assays for therapeutic compoundssuch as agonists, antagonists or inverse agonists and/or modulators formpart of the disclosure. For high-throughput purposes, compound librariesor combinatorial libraries may be used such as allosteric compoundlibraries, peptide libraries, antibody libraries, fragment-basedlibraries, synthetic compound libraries, natural compound libraries,phage-display libraries and the like. Methodologies for preparing andscreening such libraries are known to those of skill in the art.

The test compound may optionally be covalently or non-covalently linkedto a detectable label. Suitable detectable labels and techniques forattaching, using and detecting them will be clear to the skilled person,and include, but are not limited to, any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. Useful labels include magnetic beads (e.g.,dynabeads), fluorescent dyes (e.g., all Alexa Fluor dyes, fluoresceinisothiocyanate, Texas red, rhodamine, green fluorescent protein and thelike), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase), and colorimetric labelssuch as colloidal gold or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads. Means of detecting such labels arewell known to those of skill in the art. Thus, for example, radiolabelsmay be detected using photographic film or scintillation counters,fluorescent markers may be detected using a photodetector to detectemitted illumination. Enzymatic labels are typically detected byproviding the enzyme with a substrate and detecting the reaction productproduced by the action of the enzyme on the substrate, and colorimetriclabels are detected by simply visualizing the colored label. Othersuitable detectable labels were described earlier within the context ofthe first aspect of the disclosure relating to a binding domain.

Thus, according to specific embodiments, the test compound as used inany of the above screening methods is selected from the group comprisinga polypeptide, a peptide, a small molecule, a natural product, apeptidomimetic, a nucleic acid, a lipid, lipopeptide, a carbohydrate, anantibody or any fragment derived thereof, such as Fab, Fab′ and F(ab′)2,Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linkedFvs (dsFv) and fragments comprising either a VL or VH domain, a heavychain antibody (hcAb), a single domain antibody (sdAb), a minibody, thevariable domain derived from camelid heavy chain antibodies (VHH ornanobody), the variable domain of the new antigen receptors derived fromshark antibodies (VNAR), a protein scaffold including an alphabody,protein A, protein G, designed ankyrin-repeat domains (DARPins),fibronectin type III repeats, anticalins, knottins, engineered CH2domains (nanoantibodies), as defined hereinbefore.

It may be desirable to identify and characterize natural or endogenousligands of target membrane proteins. In particular, there is a need to“de-orphanise” GPCRs for which a natural activating ligand has not beenidentified. Such ligands may be recovered from biological samples suchas blood or tissue extract or from libraries of ligands. Thus, accordingto a particular embodiment, the test compound, as used in any of theabove screening methods, is provided as a biological sample. Inparticular, the sample can be any suitable sample taken from anindividual. For example, the sample may be a body fluid sample such asblood, serum, plasma, spinal fluid.

In addition to establishing binding to a target membrane protein in afunctional conformational state, it will also be desirable to determinethe functional effect of a compound on the membrane protein. Inparticular, the host cells, host cell cultures or membrane preparationsderived thereof, as described herein, can be used to screen forcompounds and/or to validate hits or leads that modulate (increase ordecrease) the biological activity of the membrane protein. The desiredmodulation in biological activity will depend on the target of choice.Taking a target GPCR as an example, the compounds may bind to the targetGPCR resulting in the modulation (activation or inhibition) of thebiological function of the GPCR, in particular the downstream receptorsignaling. This modulation of GPCR signaling can occur ortho- orallosterically. The compounds may bind to the target GPCR so as toactivate or increase receptor signaling; or alternatively, so as todecrease or inhibit receptor signaling. The compounds may also bind tothe target GPCR in such a way that they block off the constitutiveactivity of the GPCR. The compounds may also bind to the target complexin such a way that they mediate allosteric modulation (e.g., bind to theGPCR at an allosteric site). In this way, the compounds may modulate thereceptor function by binding to different regions in the GPCR (e.g., atallosteric sites). Reference is, for example, made to George et al.,2002, Nat. Rev. Drug Discov. 1:808; Kenakin 2002, Trends Pharmacol. Sci.25:186; Rios et al., 2001, Pharmacol. Ther. 92:71. The compounds of thedisclosure may also bind to the target GPCR in such a way that theyprolong the duration of the GPCR-mediated signaling or that they enhancereceptor signaling by increasing receptor-ligand affinity. Further, thecompounds may also bind to the target GPCR in such a way that theyinhibit or enhance the assembly of GPCR functional homomers orheteromers. The efficacy of the compounds and/or compositions comprisingthe same can be tested using any suitable in vitro assay, cell-basedassay, in vivo assay and/or animal model known per se, or anycombination thereof, depending on the specific disease or disorderinvolved.

It will be appreciated that the host cells and derivatives thereof,according to the disclosure, may be further engineered and are, thus,particularly useful tools for the development or improvement ofcell-based assays. Cell-based assays are critical for assessing themechanism of action of new biological targets and biological activity ofchemical compounds. For example, without the purpose of beinglimitative, current cell-based assays for GPCRs include measures ofpathway activation (Ca²⁺ release, cAMP generation or transcriptionalactivity), measurements of protein trafficking by tagging GPCRs anddownstream elements with GFP; and direct measures of interactionsbetween proteins using Fórster resonance energy transfer (FRET),bioluminescence resonance energy transfer (BRET) or yeast two-hybridapproaches.

Further, it may be particularly advantageous to immunize an animal witha host cell expressing a membrane protein and a binding domain,according to the disclosure, or a cell culture or membrane preparationderived thereof, in order to raise antibodies, preferablyconformationally-selective antibodies against the target membraneprotein. Thus, such immunization methods are also envisaged here.Methods for raising antibodies in vivo are known in the art, and arealso described hereinbefore. Any suitable animal, e.g., a warm-bloodedanimal, in particular a mammal such as a rabbit, mouse, rat, camel,sheep, cow, shark, or pig or a bird such as a chicken or turkey, may beimmunized using any of the techniques well known in the art suitable forgenerating an immune response. Following immunization, expressionlibraries encoding immunoglobulin genes, or portions thereof, expressedin bacteria, yeast, filamentous phages, ribosomes or ribosomal subunitsor other display systems, can be made according to well-known techniquesin the art. Further to that, the antibody libraries that are generatedcomprise a collection of suitable test compounds for use in any of thescreening methods, as described hereinbefore. The antibodies that havebeen raised, as described hereinabove, may also be useful diagnostictools to specifically detect membrane proteins in a particularconformational state, and, thus, also form part of the disclosure.

Still another aspect of the disclosure, relates to a kit comprising ahost cell or a host cell culture or a membrane preparation, according tothe disclosure. The kit may further comprise a combination of reagentssuch as buffers, molecular tags, vector constructs, reference samplematerial, as well as a suitable solid supports, and the like. Such a kitmay be useful for any of the applications of the disclosure, asdescribed herein. For example, the kit may comprise (a library of) testcompounds useful for compound screening applications.

The following examples are intended to promote a further understandingof the disclosure. While the disclosure is described herein, withreference to illustrated embodiments, it should be understood that thedisclosure is not limited hereto. Those having ordinary skill in the artand access to the teachings herein will recognize additionalmodifications and embodiments within the scope thereof. Therefore, thedisclosure is limited only by the claims attached herein.

EXAMPLES Co-Expression of a GPCR and an Anti-GPCR Nb in Yeast Example 1:Anti-CXCR4 Nb Cloning and Expression in Pichia pastoris Example 1.1:Pichia pastoris Cloning Procedure

Four genes that code for Nanobodies directed against the extracellularside of the human CXCR4 receptor (FIG. 1; Table 1-2) were cloned intothe pKai61 P. pastoris expression vector, in frame with a slightlymodified version of the S. cerevisiae α-mating factor signal sequence.This signal sequence directs the proteins to the yeast secretory systemand is further processed in the ER and the golgi and will be fullyremoved before secretion into the extracellular medium. In contrast tothe wild-type prepro signal, this modified version does not containsequences that code for the GluAla repeats (here the signal peptide isefficiently cleaved by the Kex2 endopeptidase without the need for thisrepeat). Expression in the secretory system is necessary for theNanobodies to come into contact with their epitopes on the hCXCR4 GPCRupon correct integration into the membranes in later co-expressionexperiments (see Example 3). The encoded genes contain a C-terminal His6tag, are N-terminally fused to the modified signal sequence of the S.cerevisiae α-mating factor and are under control of the methanolinducible AOX1 promoter. The plasmid contains a ZEOCIN® resistancemarker for selection in bacterial as well as in yeast cells. The vectorswere linearized in the AOX1 promoter (with PmeI) before transformationto P. pastoris to promote homologous recombination in the endogenousAOX1 locus for stable integration into the genome (FIG. 2).

Example 1.2: Anti-hCXCR4 Nanobody Expression in Pichia pastoris

The four anti-hCXCR4 Nanobody expression vectors were transformed to theP. pastoris GS115 strain. Stable integrants were selected on YPD platescomplemented with ZEOCIN® and confirmed by yeast colony PCR. These weregrown and induced in buffered medium, as described in Material andMethods. A fraction of the extracellular growth medium was loaded on a12% Tricine gel as such (complemented with 5× Laemmli dye and heated to95° C. for 10 minutes) and stained with Coomassie Brilliant Blue (FIG.3A) or blotted on a nitrocellulose membrane and subsequently developedwith a primary mouse anti-His monoclonal antibody and a secondaryDylight (800 nm) goat anti-mouse IgG on a LI-COR odyssey system (FIG.3B). Both the CBB-stained gel as the immunoblot showed clear proteinbands of the correct size, indicating that each Nb is expressed andsecreted by P. pastoris, without any signs of degradation.

Example 2: hCXCR4 Expression in Pichia pastoris

The full-length human CXCR4 gene was cloned into the pPIC92 P. pastorisexpression vector. The encoded gene contains a C-terminal Rho1D4-tag(TETSQVAPA), is N-terminally fused to the signal sequence of the S.cerevisiae α-mating factor and the expression is under control of themethanol inducible AOX1 promoter. The plasmid contains a HIS4 selectionmarker for selection in HIS4⁻ P. pastoris strains (e.g., GS115). Thevector is linearized in the HIS4 marker (with StuI) beforetransformation to promote homologous recombination in the endogenousHIS4 locus for stable integration into the genome (FIG. 4).

Several P. pastoris GS115 clones, of which the stable insertion of thepPIC92hCXCR4Rho1D4 vector (FIG. 4) was confirmed, were selected to checkhCXCR4 protein expression. The clones were grown and induced in bufferedmedium as described in Material and Methods. The cells were lysed andfractionated by centrifugation. Extracted proteins were tested forhCXCR4 expression by SDS-PAGE, followed by immunoblotting. Thereto,equal amounts of the different cell fractions were run on a 12% SDS-PAGEgel and blotted on a nitrocellulose membrane. The blot was developedwith a primary mouse anti-Rho1D4 monoclonal antibody and a secondaryDylight (800 nm) goat anti-mouse IgG on a LI-COR odyssey system. FIG. 5shows different cell fractions of four positive hCXCR4 expressingclones. A clear anti-Rho1D4 immunoreactive protein band is visible inthe cell lysate fraction of all clones, with only faint signals athigher molecular weight, suggesting that the protein does not have thetendency to irreversibly aggregate during expression and extraction. Thesame cell lysate fractions are subsequently centrifuged at maximum speedin a table-top centrifuge. The same protein bands can be seen in thedissolved pellet fractions, while almost no signal can be detected inthe supernatant. This indicates that the human CXCR4 receptor is indeedexpressed in the membrane protein fractions. We used clone 1 for furtherexperiments.

Example 3: hCXCR4-Nb Co-Expression in Pichia pastoris

An hCXCR4 expressing Pichia clone was transformed with the fourlinearized Nanobody expressing vectors as described before. Twenty-fourpositive clones (six per Nb) were analyzed for protein expression.Growth conditions and membrane protein sample preparations were done aspreviously described. Equal amounts of total membrane protein(determined by a BCA assay) of induced P. pastoris GS115 clones,expressing only the human CXCR4 receptor, or in combination with one ofthe anti-hCXCR4 Nanobodies, were run on a 12% SDS-PAGE gel and blottedon a nitrocellulose membrane. The blot was developed with a primarymouse anti-Rho1D4 monoclonal antibody and a secondary Dylight (800 nm)goat anti-mouse IgG on an LI-COR odyssey system. Protein expression ofthe highest hCXCR4 expressing clone of each hCXCR4-Nb combination isshown in FIG. 6. For each hCXCR4-Nb co-expressing clone we detect more(2 to 4 times) of the anti-Rho1D4 immunoreactive membrane protein bandcompared to the GS115hCXCR4 clone without Nb co-expression (except forNb4500, here we only see a slight increase). The quantification wasperformed by the Odyssey software by measuring relative fluorescentsignals (Table 3). Three out of four tested Nanobodies are able tosignificantly increase hCXCR4 protein expression in P. pastoris, asdetermined by quantifying the anti-Rho1D4 immunoreactive bands onwestern blot.

After induction, both the human CXCR4 membrane protein and theanti-hCXCR4 Nanobody are simultaneously expressed in the ER: thereceptor is integrated in the ER membrane, while the Nbs will finally besecreted into the extracellular medium, unless they are able to bindtheir epitopes on the receptor somewhere along the secretory system. Tocheck for possible in vivo binding, we first analyzed the intracellularretention of the Nanobodies in cells expressing Nbs alone or incombination with the human CXCR4 receptor (FIG. 7). Equal membraneprotein fractions of induced P. pastoris GS115 clones, expressing onlythe human CXCR4 receptor or one of the four anti-hCXCR4 Nanobodies, or acombination, were run on a 15% SDS-PAGE gel and blotted on anitrocellulose membrane. The blot was developed with a primary mouseanti-His monoclonal antibody and a secondary Dylight (800 nm) goatanti-mouse IgG on an LI-COR odyssey system. All Nanobodies are clearlyretained in the membrane protein fractions of hCXCR4-Nb co-expressingclones (protein band around ±15 kDa), while almost no signal is detectedin protein samples of clones lacking the hCXCR4 receptor. The extra bandaround 25 kDa represents a not fully processed form of the Nanobody,i.e., a form in which part of the signal peptide of the S. cerevisiaealpha-mating factor is yet uncleaved.

In order to demonstrate that the increase in receptor expression is dueto the interaction of a Nanobody specific for the receptor, a controlexperiment was performed using a Nanobody which does not recognize thehCXCR4 (Nanobody CA4910; amino acid sequence: SEQ ID NO:6; Nucleotidesequence: SEQ ID NO:24). An hCXCR4 expressing Pichia clone wastransformed with the linearized Nanobody expressing vector as describedbefore. Three positive clones were analyzed for protein expression.Growth conditions and membrane protein sample preparations were done aspreviously described. Equal amounts of total membrane protein(determined by a BCA assay) of induced P. pastoris GS115 clones,expressing only the human CXCR4 receptor, or in combination with aanti-hCXCR4 Nanobody (NbCA4142), or in combination with the negativecontrol Nanobody (NbCA4910), were run on a 12% SDS-PAGE gel and blottedon a nitrocellulose membrane. The blot was developed with a primarymouse anti-Rho1D4 monoclonal antibody and a secondary Dylight (800 nm)goat anti-mouse IgG on an LI-COR odyssey system. Protein expression ofthe hCXCR4 is shown in FIG. 8. For the hCXCR4-specific Nb co-expressingclone we detect more of the anti-Rho1D4 immunoreactive membrane proteinband compared to the GS115hCXCR4 clone without Nb co-expression. For thethree clones, co-expressing the Nanobody that does not recognize thereceptor, it is clear that there is no increase in the receptorexpression. Exact quantification has been calculated by the Odysseysoftware by measuring relative fluorescent signals (Table 4). It can,thus, be concluded that the increase in receptor expression can beattributed to the Nanobody-specific interaction with the receptor.

Example 4: hCXCR4 Purification from P. pastoris Based on the NanobodyInteraction

Because there is a strong interaction between the hCXCR4 receptor andNbCA4142, we could purify the membrane protein-Nanobody complex from aco-expressing clone, by Ni²⁺ affinity chromatography for thehexaHis-tagged Nanobody. In this way we only purify the receptorfraction that is in a well-folded state, because the Nanobody can onlybind the receptor when it is in its native fold. This step stronglyincreases homogeneity in a membrane protein sample which is useful, forexample, for crystallization. Membrane protein preparation andpurification was performed, as described in the methods section, andclearly resulted in strong retention of the receptor when brought on aNi²⁺ affinity column (FIG. 9). In case an even more pure sample isrequired, an extra step of affinity chromatography can be performedusing an anti-Rho1D4 affinity matrix, which targets the hCXCR4 part ofthe complex.

Co-Expression of a GPCR and an Anti-GPCR Nb in Mammalian Cells Example5: Co-Expression of β2AR-Nanobody in HEK293S GnT⁻ Cells and Purificationof β2AR Based on the Nanobody Interaction

The same technique as in Example 4 can be used using a Nanobody thatbinds an intracellular epitope of the membrane protein. Nanobody CA2780(amino acid sequence: SEQ ID NO:5; Nucleotide sequence: SEQ ID NO:23) isan example of a Nanobody that binds an intracellular epitope of theβ2-adrenergic receptor and that stabilizes the receptor in its activeconformation (Rasmussen et al., 2011, Nature 469: 175). We transformed aHEK293 cell line that has a stable inducible β2AR expression with thepCAGGSNbCA2780-plasmid (see Material and Methods section) for transientexpression. This Nanobody is not fused to a secretion signal and,therefore, resides in the cytosol upon expression, to allow interactionwith the intracellular side of the membrane protein. Nanobody CA2780 hasa hexaHis-tag and can, therefore, be purified using Ni²⁺ affinitychromatography. Because there is a strong interaction between the β2ARreceptor and NbCA2780 upon the addition of the agonist alprenololhydrochloride, we could purify the membrane protein-Nanobody complex byNi²⁺ affinity chromatography for the hexaHis-tag on the Nanobody partnerin the complex (FIG. 10). In this way we strongly enrich for thereceptor fraction that is in a well-folded state, because the Nanobodycan only bind the receptor when it is in its native fold. This stepstrongly increases homogeneity in a membrane protein sample going tof.e. crystallization trials. If necessary, a subsequent purificationusing anti-Rho1D4 affinity chromatography can further enrich the samplefor the receptor.

Material and Methods

Materials

The P. pastoris GS115 strain was used for all yeast expressionexperiments. The pPIC92 vector was originally obtained from Invitrogen.The origin of the pKai61 vector is described in Schoonooghe et al.,2009, BMC Biotechnol. 9:70. MC1061 cells were used for the amplificationof recombinant plasmid DNA.

The pCAGGS vector is described in Niwa et al., 1991, Gene, 108, 2:193-199, and on the World Wide Web at addgene.org/vector-database/2042/,and was obtained from the BCCM plasmid collection. The HEK293S GnT⁻ cellline was obtained from the Prof. Dr. Prashen Chelikani (University ofManitoba, Winnipeg, Canada).

Media

Depending on the experimental settings, yeast strains were grown in YPDmedium (10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose, ±20 g/Lagar), BMY buffered complex medium (10 g/L yeast extract, 20 g/Lpeptone, 100 mM potassium phosphate buffer pH 6.0, 13.4 g/L YNB withoutamino acids, complemented with 1% glycerol (BMGY) or 1% methanol(BMMY)), Minimal medium without histidine (MM-HIS; 20 g/L agar, 13.4 g/LYNB without amino acids, 20 g/L dextrose, 0.79 g/L CSM-HIS).

Cloning

Construction of pKai61-antihCXCR4Nb Plasmid

All four Nb genes were PCR amplified (using Phusion polymerase) from thepMES4 vector in which they were initially cloned using primers NbXhoIFw5′-GAAGAAGGGGTATCTCTCGAGAAAAGGCAGGTGCAGCTGCAGG-3′ (SEQ ID NO:26) andNbSpeIRev 5′-TCATGTCTAAGGCTAACTAGTCTAGTGATGGTGATGG TGG-3′ (SEQ IDNO:27). The Nanobody specific sequence is shown in italic; the 5′portion of each primer contains an XhoI and a SpeI RE recognition site,respectively, preceded by 15 bp homology to the pKai61 destinationvector. The Fw primer also contains extra nucleotides to complete thesignal sequence cleavage site. Each gel purified Nb PCR fragment wascloned into the linearized (XhoI and SpeI, fermentas; gel purified)pKai61 destination vector (in frame with the alpha-mating factor signalsequence from S. cerevisiae and under control of the methanol inducibleAOX1 promoter; the vector also contains a ZEOCIN® resistance cassettefor bacterial and yeast selection) using the commercial CloneEZrecombination kit, according to the manufacturer's instructions(Genscript). The reaction mixtures were transformed to E. coli MC1061competent cells and ZEOCIN®-resistant clones were selected on LB LowSalt plates containing 50 μg/ml ZEOCIN®. Transformants were screened bycolony PCR using the 5′ (5′-GACTGGTTCCAATTGACAAGC-3′; SEQ ID NO:28) and3′ (5′-GCAAATGGCATTCTGACATCC-3′; SEQ ID NO:29) AOX1 promoter andterminator primers. All four final pKai61-antihCXCR4Nb plasmids wereexamined by restriction enzyme digestion and the inserts were sequenceverified with the same 5′ and 3′ AOX1 primers.

Construction of pPIC92hCXCR4Rho1D4 Plasmid

The human CXCR4 gene (GenBank gene ID 7852, isoform a), fused to asequence coding for the C-terminal Rho1D4 tag (TETSQVAPA; SEQ ID NO:25),was codon optimized for P. pastoris and synthetically synthesized by theGenscript corporation (cloned and delivered in the standard pUC57vector). The gene was cloned into the pPIC92 P. pastoris expressionvector, in frame with the α-mating factor signal sequence of S.cerevisiae, using conventional RE cloning (XhoI and NotI, fermentas).The ligation mixture was transformed to E. coli MC1061 competent cellsand carbenicillin resistant clones were selected on LB Low Salt platescontaining 50 μg/ml carbenicillin. Transformants were screened by colonyPCR using the 5′ and 3′ AOX1 promoter and terminator primers. The finalpPIC92hCXCR4Rho1D4 plasmid was examined by RE digestion and thehCXCR4Rho1D4 insert was sequence verified using the 5′ and 3′ AOX1primers. The membrane protein gene is under control of the methanolinducible AOX1 promoter and the vector also contains a HIS4 marker forauxotrophic selection in the P. pastoris GS115 strain (HIS4⁻).

Yeast Transformation and Screening

The pPIC92hCXCR4Rho1D4 and the pKai61-antihCXCR4Nb plasmids werelinearized (in the HIS4 resistance marker using StuI or in the AOX1promoter region using PmeI, respectively) to increase transformationefficiency and to promote homologous recombination in the endogenousHIS4 or AOX1 locus for stable integration of the vector into the genome.Competent P. pastoris GS115 (HIS4⁻) cells were prepared and transformedby electroporation, according to the protocol from the Pichia Expressionkit (Invitrogen Cat. No. K1710-01). Linearized plasmid (1 μg) was mixedwith 80 μl competent cells in a 0.2 cm electroporation cuvette and themixture was pulsed, according to the following settings: 25 ρF; 200 Ω;1.5 kV. Transformants were plated on selective medium (Minimal mediumwithout histidine or YPD plates containing 100 μg/ml ZEOCIN®). Pichiacolony PCR (i.e., yeast cells were pre-treated before the PCR reactionwith a commercial zymolyase enzyme mixture and snap-frozen to weaken thecell wall) was performed using the 5′ and 3′ AOX1 primers to confirm thestable integration of the genes into the P. pastoris genome.

Yeast Cell Culture Conditions

Stable positive clones were typically grown in 2 ml (24-well format) or12.5 ml (shake-flasks) BMGY medium for 48 hours (250 rpm; 28° C.). Thecells were harvested by centrifugation and resuspended in the sameamount of BMMY medium (1% methanol; 0.5% of methanol was added after 12hours to maintain induction). After 24 hours the cells were harvestedfor membrane protein preparations (analysis of hCXCR4 expression andintracellular Nb retention) and the supernatant was used for analysis ofextracellular protein expression on SDS-PAGE (Nanobodies).

Membrane Protein Preparations

The cell pellet corresponding to 1 ml of saturated yeast culture (OD₆₀₀60-80) was suspended in 1 ml of PBS+Protease inhibitor (Roche). Togetherwith 200 μl of acid washed glass beads, the samples were vortexedheavily at 4° C. for 2×5 minutes. Glass beads, cell debris and intactcells were removed by centrifugation at 2000 rpm (5 min; 4° C.). Thesupernatant was kept as such or was centrifuged for 45 minutes at 13,000rpm in a desktop centrifuge at 4° C. The supernatant, cytosolic fractionwas discarded and the pellet fraction was re-dissolved in the sameamount of fresh PBS+PI by sonication until a homogenous sample wasobtained.

Total membrane protein concentration was determined by the BCA proteinassay kit (Pierce, Thermo Scientific).

SDS-PAGE and Western Blot Analysis

Protein samples were analyzed by SDS-poly-acrylamide gelelectrophoresis, according to the Laemmli (Laemmli et al., 1970, Nature,227:680) or the Tricine method (Schägger et al., 2006, Nat. Protoc.1:16-22). Before loading on gel, the samples were mixed with 5× Laemmli(60 mM Tris-HCl pH 6.8; 2% SDS, 10% glycerol, 5% β-ME; 0.01% bromophenolblue) and heated for 10 minutes at 95° C. (only the ECM samples, not themembrane protein preparations). Prestained protein markers in the rangeof 10-250 kDa, Precision Plus protein standards (Bio-Rad), were used asmolecular standards. Proteins in the gel were stained with CoomassieBrilliant Blue R-350 or they were electrotransferred at 10V, 100 mA/gel(semi-dry) for 90 minutes onto nitrocellulose membranes. The membraneswere blocked overnight (4° C.) in PBS TWEEN® 20 (0.05%) containing 5%milk powder. They were then incubated for one hour at room temperaturewith a mouse anti-His (1/1000 of a 0.2 mg/ml stock in PBST; detection ofNanobodies) or a mouse anti-Rho1D4 (1/500 of a 5 mg/ml stock in PBST;detection of hCXCR4) monoclonal antibody, washed 3 times with PBST andthen incubated for one hour with a secondary goat anti-mouse antibody(Dylight 800 nm). After the final washing steps, the membranes werescanned with a LI-COR Odyssey system. Analysis and quantification of thesignals were done with the Odyssey software.

Construction of pCAGGSNbCA2780 Plasmid

The Nanobody gene was PCR amplified (using Phusion polymerase) from thepMES4 vector in which it was originally cloned using primers Nb80Fw Mc5′-agtctgCTCGAGCCACCATGGAGGTGCAGCTGCAGG-3′ (SEQ ID NO:30) and Nb80Rev Mc5′-cagactAGATCTctaGTGATGGTGATGATGATGTGCGGCCGCTGAGGAGACGGTGA CCTGGGTCC-3′(SEQ ID NO:31). The Nanobody specific sequence is shown in italic; the5′ portion of each primer contains an XhoI and a BglII RE recognitionsite, respectively. Both primers also contain extra nucleotides toenable a restriction digest on the PCR product. The gel purified Nb PCRfragment was cloned into the linearized (XhoI (Fermentas), BglII(Promega); gel purified) pCAGGS destination vector, under control of theCMV immediate early promoter using T4 ligase (Fermentas); the vectoralso contains an ampicillin/carbenicillin resistance gene for bacterialselection. The reaction mixture was transformed to E. coli MC1061competent cells and carbenicillin-resistant clones were selected on LBLow Salt plates containing 50 μg/ml carbenicillin. The finalpCAGGSNbCA2780 plasmid was examined by restriction enzyme digestion andthe inserts were sequence verified with the traditional pCAGGSsequencing primers.

Transfection and Induction of NbCA2780 in HEK293S-β2AR

HEK293S-β2AR expressing cells (Chelikani et al., 2006, Protein Sci15:1433) were grown to 60% to 80% confluence in a T175 Falcon andtransfected with 1 μg pCAGGSNbCA2780 using FuGeneHD Transfection Reagent(Promega). After 12 h, induction was performed using 1 μg/ml doxycyclin,7.5 mM Na-butyrate and 1 μM alprenolol hydrochloride for 36 h.

Membrane Protein Isolation from P. pastoris for Purification

The cell pellet corresponding to 1 L of saturated yeast culture (OD₆₀₀60-80) was resuspended in a 1:1 (w/v) ratio of buffer A (1×PBS+Proteaseinhibitors). Together with 190 g of glass beads, the suspension wasbrought in the precooled DYNO®-MILL mixing chamber and cells werecrushed for 5 minutes. The lysate was centrifuged for 10 minutes at2,000 rpm (Sorvall F10S-6X500Y rotor) at 4° C. to remove cell debris andintact cells. The supernatant was centrifuged for an hour at 13,000 rpm(Sorvall F10S-6X500Y rotor) at 4° C. to pellet the membrane proteins.The pellet was resuspended in 10 ml buffer A with 1% DDM (n-Dodecylβ-D-maltoside) and put on a rotor for three hours at 4° C. to allowmembrane protein solubilization. After solubilization, the solution iscentrifuged for one hour at 13,000 rpm (Sorvall SS-34 rotor) at 4° C. toremove unsolubilized membrane proteins.

Membrane Protein Isolation from HEK293S GnT⁻ for Purification

The cell pellet corresponding to one confluent T175 Falcon oftransfected β2AR-expressing cells with NbCA2780 was resuspended in 4 mLlysis/solubilization buffer (1% NP40, 200 mM NaCl, 10 mM TrisHCl pH 7.5,5 mM EDTA, 10% Glycerol, Protease Inhibitors, 1% DDM) and allowed torotate for one hour at 4° C. The lysate is centrifuged at 2,000 rpm for5 minutes to remove cell debris. The supernatant now contains thesolubilized membrane proteins as well as the cytosol.

Membrane Protein Purification Using Ni²⁺ Affinity Chromatography

The solubilized MP fraction is added to 0.5 ml of Ni²⁺-loaded ChelatingSepharose Fast Flow matrix. Binding is performed overnight on a rotor at4° C. Four consecutive washes are performed using 10 ml of buffer B (20mM NaH₂PO₄, 500 mM NaCl, 20 mM Imidazole at pH 7.5). Then fourconsecutive elutions are done using increasing concentrations of bufferC (20 mM NaH₂PO₄, 20 mM NaCl, 400 mM Imidazole at pH 7.5). For theanalysis, equal volumes were loaded on a 12% SDS-PAGE gel and Westernblot was performed, as described before.

TABLE 1 List of nanobodies Nanobody reference number SEQ ID NOAmino acid sequence (including HIS tag) CXCR4- specific nanobodiesCA4140 1 QVQLQESGGGLVQPGGSLRLSCAASGSIFSINAMGWYRQAPGKQRELVAAITSGGSTNYADSVKGRFTISRDNAENTVYLQMNNLKPEDTAVYSCNAEGTSGSSRYRRRYEYWG KGTQVTVSSHHHHHH CA4142 2QVQLQESGGGLVRTGGSLRLSCAGSGSFFSINPMGWYRQAPGQQRELVATITGSGSTNYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCNAGYFDRIGRRYDRWGQGT QVTVSSHHHHHH CA4143 3QVQLQESGGGLYQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTINSGGRSANYADSVKGRLTISRDNAKNTLHLQMNSLKPEDTALYYCARPRSVSRNYVPLGYDYL GQGTQVTVSSHHHHHH CA4500 4QVQLQESGGGLVQAGGSLRLSGAASGSTSGIIAMGWYRQAPGKQRELVARISSGSSTNYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCNAVRRGYRNDYNSWGQG TQVTVSSHHHHHH B2AR- specificnanobodies CA2780 5 MEVQLQESGGGLVQAGGSLRLSCAASGSIFSINTMGWYRQAPGKQRELVAAIHSGGSTNYANSVKGRFTISRDNAANTVYLQMNSLKPEDTAVYYCNVKDYGAVLYEYDYWG QGTQVTVSSAAAHHHHHH control nanobodyCA4910 6 QVQLVESGGGLVQPGGSLRLSCAASGSFRSIVSMAWYRQAPGKQRELVASSNSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYWCNVQNRLPGFDAFSGRSIAE TYWGQGTQVTVSSAAAHHHHHH CXCR4-specific nanobodies CA4140 19caggtgcagctgcaggagtctgggggaggcttggtgcagcctggggggtctctgagactctcctgtgcagcctctggaagcatcttcagtatcaatgccatgggctggtaccgccaggctccagggaagcagcgcgagttggtcgcagctattactagtggtggtagcacaaactatgcagactccgtaaagggccgattcaccatctccagagacaacgccgagaacacggtgtatctgcaaatgaacaacctgaaacctgaggacacggccgtctattcatgtaacgctgaaggaacgtcgggtagtagccggtatcgccgccggtatgagtactggggcaaggggacccaggtcaccgtctcctcacaccaccatcaccatcac CA4142 20caggtgcagctgcaggagtctggaggaggcttggtgcgcactggggggtctagagactctcctgtgaggctctggaagcttcttcagtatcaatcccatgggctggtaccgccaggctccagggcagcagcgcgagttggtcgcaactattactggtagtggtagcacaaactatgcagactccgtgaagggccgattcaccatctccagagacaacgccaagaacacactgtatctgcaaatgaacagcctgaaacctgaggacacggccgtctattactgtaatgcaggatatttcgatcggattggtcggcggtatgaccgctggggccaggggacccaggtcaccgtctcctcacaccaccatcaccatcac CA4143 21caggtgcagctgcaggagtctggaggaggcttggtgcagcctggggggtctctgagactctcctgtgcagcctctggattcaccttcagtagctatgccatgagctgggtccgccaggctccaggaaaggggctcgagtgggtctcaactattaatagtggtggtcgtagcgcaaactatgcagactccgtgaagggccgactcaccatctccagagacaacgccaagaacacgctgcatctgcaaatgaacagcctgaaacctgaggacacggccctgtattactgtgcgagaccccgtagtgtaagtcgcaactatgttccactcggatacgactacttgggccaggggacccaggtcaccgtctcctcacaccaccatcaccatcactag CA4500 22caggtgcagctgcaggagtctggaggaggcttggtgcaggctggggggtctctgagactctcctgtgagcctctggaagcacctccggtatcattgccatgggctggtaccgccaggctccagggaagcagcgcgagttggtcgcacgtattagtagtggtagtagtacaaactatgcagactccgtgaagggccgattcaccgtctccagagacaacgccaagaacacagtgtatctgcaaatgaacagcctgaaacctgaggacacggccgtctattactgtaatgcagtccgtcgaggttaccgtaacgactataactcctggggccaggggacccaggtcaccgtctcctcacaccaccatcaccatcactagactagt β2AR specific nanobodies CA2780 23atggaggtgcagctgcaggagtctgggggaggcttggtgcaggctggggggtctctgagactctcctgtgcagcctctggaagcatcttcagtatcaataccatgggctggtaccgccaggctccagggaagcagcgcgagttggtcgcagctattcatagtggtggtagcacaaactatgccaactccgtgaagggccgattcaccatctccagagacaatgccgcgaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccgtctattactgtaatgtaaaggactacggggcggtcctatatgagtatgactactggggccaggggacccaggtcaccgtctcctcagcggccgcacatcatcatcaccatcactag control nanobody CA4910 24caggtgcagctggtggagtctgggggaggcttggtgcagcctggggggtctctgagactctcctgtgcagcctctggaagcttccgcagtatcgtgtctatggcctggtaccgccaggctccagggaagcagcgcgagttggtcgcaagttctaatagtgggggcagcacaaattatgcagactccgtgaagggccgattcaccatctccagagacaacgccaagaacacggtgtatctgcaaatgaacagcctgaaacctgaggacacggccgtgtattggtgtaatgtccaaaaccgcctcccgggattcgacgcctttagtggcagatctatagcggagacctattggggccaggggacccaggtcaccgtctcctcagcggccgcacatcatcatcaccatcac

TABLE 2 CDRs of CXCR4-specific nanobodies Nanobody reference CDR1 CDR2CDR3 number (SEQ ID NO) (SEQ ID NO) (SEQ ID NO) CA4140 GSIFSINAAAITSGGSTNYADSVK NAEGTSGSSRYRRRYEY (SEQ ID NO: 7) (SEQ ID NO: 8)(SEQ ID NO: 9) CA4142 GSFFSINP ATITGSGSTNYADSVK NAGYFDRIGRRYDR(SEQ ID NO: 10) (SEQ ID NO: 11) (SEQ ID NO: 12) CA4143 GFTTFSSYAINSGGRSANYADSVK ARPRSVSRNYVPLGYDY (SEQ ID NO: 13) (SEQ ID NO: 14)(SEQ ID NO: 15) CA4500 GSTSGIIA ARISSGSSTNYADSVK NAVRRGYRNDYNS(SEQ ID NO: 16) (SEQ ID NO: 17) (SEQ ID NO: 18)

TABLE 3 Relative hCXCR4 expression improvement factor for hCXCR4-Nbco-expressing strains versus the hCXCR4 expressing strain FluorescentBlank Factor Sample units corrected improvement GS115 Negative control3.34 0 — GS115 + hCXCR4Rho1D4 16.81 13.47 1.0 (CL1) GS115 + hCXCR4Rho1D439.95 36.61 2.8 (CL1) + NbCA4140 GS115 + hCXCR4Rho1D4 35.81 32.47 2.6(CL1) + NbCA4142 GS115 + hCXCR4Rho1D4 41.06 37.72 3.0 (CL1) +NbCA4143GS115 + hCXCR4Rho1D4 21.59 18.25 1.3 (CL1) + NbCA4500

The shown relative fluorescent units are calculated by averaging data oftwo independent experiments. The “fluorescent unit” values are measuredby the odyssey software.

TABLE 4 Relative hCXCR4 expression improvement factor for hCXCR4-Nbco-expressing strain versus the hCXCR4 expressing strain and thehCXCR4-Nb4910 co-expression strain Fluorescent Blank Factor Sample unitscorrected improvement GS115 Negative control 2.41 0 — GS115 +hCXCR4Rho1D4 18.45 16.04 1.0 (CL1) GS115 + hCXCR4Rho1D4 32.76 30.35 1.9(CL1) + NbCA4142 GS115 + hCXCR4Rho1D4 8.97 6.56 0.4 (CL1) + NbCA4910 cl1GS115 + hCXCR4Rho1D4 20.30 17.89 1.1 (CL1) + NbCA4910 cl2 GS115 +hCXCR4Rho1D4 21.90 19.49 1.2 (CL1) + NbCA4910 cl3 The “fluorescent unit”values are measured by the odyssey software.

What is claimed is:
 1. A recombinant cell comprising: a first exogenouspolynucleotide encoding a GPCR, and a second exogenous polynucleotideencoding an immunoglobulin single variable domain that specificallybinds an intracellular conformational epitope of the GPCR, wherein theexpression of each exogenous polynucleotide is under the control of apromoter; wherein the amount of the GPCR in the recombinant cell isincreased by at least two-fold as compared to an otherwise identicalcontrol cell not comprising the second exogenous polynucleotide; whereinthe immunoglobulin single variable domain was identified by a methodcomprising: detecting specific binding of the immunoglobulin singlevariable domain to an intracellular conformational epitope of the GPCR;co-expressing the immunoglobulin single variable domain with the GPCR ina host cell; and detecting an increased amount of the GPCR by at leasttwo-fold in the host cell as compared to an otherwise identical hostcell not comprising the immunoglobulin single variable domain.
 2. Therecombinant cell of claim 1, wherein detecting specific binding of theimmunoglobulin single variable domain to the GPCR comprises screening alibrary of immunoglobulin single variable domains to identify animmunoglobulin single variable domain that, when coexpressed with theGPCR, specifically binds to an intracellular conformational epitope inthe GPCR, and increases the amount of the GPCR in the recombinant cellas compared to an otherwise identical cell not comprising theimmunoglobulin single variable domain.
 3. The recombinant cell of claim2, wherein screening a library of immunoglobulin single variable domainsto identify an immunoglobulin single variable domain comprisesidentifying an immunoglobulin single variable domain that increases theamount of the GPCR in the cell by at least two-fold as compared to anotherwise identical cell not comprising the immunoglobulin singlevariable domain.
 4. The recombinant cell of claim 1, wherein saidpromoter is a constitutive promoter or an inducible promoter.
 5. Therecombinant cell of claim 1, wherein said GPCR and immunoglobulin singlevariable domain are co-expressed by the recombinant cell.
 6. Therecombinant cell of claim 1, wherein said GPCR and/or saidimmunoglobulin single variable domain are operably linked to one or moresubcellular targeting sequences.
 7. The recombinant cell of claim 1,wherein said immunoglobulin single variable domain comprises a peptidecomprising four framework regions and three complementary determiningregions, or any suitable fragment thereof.
 8. The recombinant cellaccording to claim 7 wherein said immunoglobulin single variable domainis a nanobody.
 9. The recombinant cell of claim 1, wherein saidimmunoglobulin single variable domain stabilizes said GPCR in afunctional conformational state.
 10. The recombinant cell of claim 1,wherein said recombinant cell is a eukaryotic cell.
 11. The recombinantcell of claim 10 wherein said recombinant cell is a yeast selected fromthe group consisting of a Pichia strain, a Komagataella strain, aHansenula strain, a Yarrowia strain, and a Saccharomyces strain.
 12. Therecombinant cell according to claim 10, wherein said recombinant cell isof human origin.
 13. The recombinant cell of claim 10, wherein saidrecombinant cell is an Sf9 cell.
 14. The recombinant cell of claim 1,wherein said recombinant cell is a glycoengineered cell.
 15. Therecombinant cell of claim 10, wherein the recombinant cell is afilamentous fungi selected from the group consisting of an Aspergillusstrain, a Penicillium strain, and a Hypocrea strain.